Invasive cleavage of nucleic acids

ABSTRACT

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

The present application is a Continuation of U.S. application Ser. No.09/350,309, filed Jul. 9, 1999, now U.S. Pat. No. 6,348,314, which is aDivisional of U.S. application Ser. No. 08/756,386, filed Nov. 26, 1996,now U.S. Pat. No. 5,985,557, which is a Continuation-In-Part of U.S.application Ser. No. 08/682,853, filed Jul. 12, 1996, now U.S. Pat. No.6,001,567, which is a Continuation-In-Part of U.S. application Ser. No.08/599,491, filed Jan. 24, 1996, now U.S. Pat. No. 5,846,717.

FIELD OF THE INVENTION

The present invention relates to means for the detection andcharacterization of nucleic acid sequences and variations in nucleicacid sequences. The present invention relates to methods for forming anucleic acid cleavage structure on a target sequence and cleaving thenucleic acid cleavage structure in a site-specific manner. The 5′nuclease activity of a variety of enzymes is used to cleave thetarget-dependent cleavage structure, thereby indicating the presence ofspecific nucleic acid sequences or specific variations thereof. Thepresent invention further provides novel methods and devices for theseparation of nucleic acid molecules based by charge.

BACKGROUND OF THE INVENTION

The detection and characterization of specific nucleic acid sequencesand sequence variations has been utilized to detect the presence ofviral or bacterial nucleic acid sequences indicative of an infection,the presence of variants or alleles of mammalian genes associated withdisease and cancers and the identification of the source of nucleicacids found in forensic samples, as well as in paternity determinations.

Various methods are known to the art which may be used to detect andcharacterize specific nucleic acid sequences and sequence variants.Nonetheless, as nucleic acid sequence data of the human genome, as wellas the genomes of pathogenic organisms accumulates, the demand for fast,reliable, cost-effective and user-friendly tests for the detection ofspecific nucleic acid sequences continues to grow. Importantly, thesetests must be able to create a detectable signal from samples whichcontain very few copies of the sequence of interest. The followingdiscussion examines two levels of nucleic acid detection assayscurrently in use: I. Signal Amplification Technology for detection ofrare sequences; and II. Direct Detection Technology for detection ofhigher copy number sequences.

I. Signal Amplification Technology Methods For Amplification

The “Polymerase Chain Reaction” (PCR) comprises the first generation ofmethods for nucleic acid amplification. However, several other methodshave been developed that employ the same basis of specificity, butcreate signal by different amplification mechanisms. These methodsinclude the “Ligase Chain Reaction” (LCR), “Self-Sustained SyntheticReaction” (3SR/NASBA), and “Qβ-Replicase” (Qβ).

Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR), as described in U.S. Pat. Nos.4,683,195 and 4,683,202 to Mullis and Mullis et al. (the disclosures ofwhich are hereby incorporated by reference), describe a method forincreasing the concentration of a segment of target sequence in amixture of genomic DNA without cloning or purification. This technologyprovides one approach to the problems of low target sequenceconcentration. PCR can be used to directly increase the concentration ofthe target to an easily detectable level. This process for amplifyingthe target sequence involves introducing a molar excess of twooligonucleotide primers which are complementary to their respectivestrands of the double-stranded target sequence to the DNA mixturecontaining the desired target sequence. The mixture is denatured andthen allowed to hybridize. Following hybridization, the primers areextended with polymerase so as to form complementary strands. The stepsof denaturation, hybridization, and polymerase extension can be repeatedas often as needed, in order to obtain relatively high concentrations ofa segment of the desired target sequence.

The length of the segment of the desired target sequence is determinedby the relative positions of the primers with respect to each other,and, therefore, this length is a controllable parameter. Because thedesired segments of the target sequence become the dominant sequences(in terms of concentration) in the mixture, they are said to be“PCR-amplified.”

Ligase Chain Reaction (LCR or LAR)

The ligase chain reaction (LCR; sometimes referred to as “LigaseAmplification Reaction” (LAR) described by Barany, Proc. Natl. Acad.Sci., 88:189 (1991); Barany, PCR Methods and Applic., 1:5 (1991); and Wuand Wallace, Genomics 4:560 (1989) has developed into a well-recognizedalternative method for amplifying nucleic acids. In LCR, fouroligonucleotides, two adjacent oligonucleotides which uniquely hybridizeto one strand of target DNA, and a complementary set of adjacentoligonucleotides, which hybridize to the opposite strand are mixed andDNA ligase is added to the mixture. Provided that there is completecomplementarity at the junction, ligase will covalently link each set ofhybridized molecules. Importantly, in LCR, two probes are ligatedtogether only when they base-pair with sequences in the target sample,without gaps or mismatches. Repeated cycles of denaturation,hybridization and ligation amplify a short segment of DNA. LCR has alsobeen used in combination with PCR to achieve enhanced detection ofsingle-base changes. Segev, PCT Public. No. W09001069 A1 (1990).However, because the four oligonucleotides used in this assay can pairto form two short ligatable fragments, there is the potential for thegeneration of target-independent background signal. The use of LCR formutant screening is limited to the examination of specific nucleic acidpositions.

Self-Sustained Synthetic Reaction (3SR/NASBA)

The self-sustained sequence replication reaction (3SR) (Guatelli et al.,Proc. Natl. Acad. Sci., 87:1874-1878 [1990], with an erratum at Proc.Natl. Acad. Sci., 87:7797 [1990]) is a transcription-based in vitroamplification system (Kwok et al., Proc. Natl. Acad. Sci., 86:1173-1177[1989]) that can exponentially amplify RNA sequences at a uniformtemperature. The amplified RNA can then be utilized for mutationdetection (Fahy et al., PCR Meth. Appl., 1:25-33 [1991]). In thismethod, an oligonucleotide primer is used to add a phage RNA polymerasepromoter to the 5′ end of the sequence of interest. In a cocktail ofenzymes and substrates that includes a second primer, reversetranscriptase, RNase H, RNA polymerase and ribo-and deoxyribonucleosidetriphosphates, the target sequence undergoes repeated rounds oftranscription, cDNA synthesis and second-strand synthesis to amplify thearea of interest. The use of 3SR to detect mutations is kineticallylimited to screening small segments of DNA (e.g., 200-300 base pairs).

Q-Beta (Qβ) Replicase

In this method, a probe which recognizes the sequence of interest isattached to the replicatable RNA template for Qβ replicase. A previouslyidentified major problem with false positives resulting from thereplication of unhybridized probes has been addressed through use of asequence-specific ligation step. However, available thermostable DNAligases are not effective on this RNA substrate, so the ligation must beperformed by T4 DNA ligase at low temperatures (37° C.). This preventsthe use of high temperature as a means of achieving specificity as inthe LCR, the ligation event can be used to detect a mutation at thejunction site, but not elsewhere.

Table 1 below, lists some of the features desirable for systems usefulin sensitive nucleic acid diagnostics, and summarizes the abilities ofeach of the major amplification methods (See also, Landgren, Trends inGenetics 9:199 [1993]).

A successful diagnostic method must be very specific. A straight-forwardmethod of controlling the specificity of nucleic acid hybridization isby controlling the temperature of the reaction. While the 3SR/NASBA, andQβ systems are all able to generate a large quantity of signal, one ormore of the enzymes involved in each cannot be used at high temperature(i.e., >55° C.). Therefore the reaction temperatures cannot be raised toprevent non-specific hybridization of the probes. If probes areshortened in order to make them melt more easily at low temperatures,the likelihood of having more than one perfect match in a complex genomeincreases. For these reasons, PCR and LCR currently dominate theresearch field in detection technologies.

TABLE 1 METHOD: PCR & 3SR FEATURE PCR LCR LCR NASBA Qβ AmplifiesTarget + + + + Recognition of Independent + + + + + Sequences RequiredPerformed at High Temp. + + Operates at Fixed Temp. + + ExponentialAmplification + + + + + Generic Signal Generation + Easily Automatable

The basis of the amplification procedure in the PCR and LCR is the factthat the products of one cycle become usable templates in all subsequentcycles, consequently doubling the population with each cycle. The finalyield of any such doubling system can be expressed as: (1+X)^(n)=y,where “X” is the mean efficiency (percent copied in each cycle), “n” isthe number of cycles, and “y” is the overall efficiency, or yield of thereaction (Mullis, PCR Methods Applic., 1:1 [1991]). If every copy of atarget DNA is utilized as a template in every cycle of a polymerasechain reaction, then the mean efficiency is 100%. If 20 cycles of PCRare performed, then the yield will be 2²⁰ , or 1,048,576 copies of thestarting material. If the reaction conditions reduce the mean efficiencyto 85%, then the yield in those 20 cycles will be only 1.85²⁰, or220,513 copies of the starting material. In other words, a PCR runningat 85% efficiency will yield only 21% as much final product, compared toa reaction running at 100% efficiency. A reaction that is reduced to 50%mean efficiency will yield less than 1% of the possible product.

In practice, routine polymerase chain reactions rarely achieve thetheoretical maximum yield, and PCRs are usually run for more than 20cycles to compensate for the lower yield. At 50% mean efficiency, itwould take 34 cycles to achieve the million-fold amplificationtheoretically possible in 20, and at lower efficiencies, the number ofcycles required becomes prohibitive. In addition, any backgroundproducts that amplify with a better mean efficiency than the intendedtarget will become the dominant products.

Also, many variables can influence the mean efficiency of PCR, includingtarget DNA length and secondary structure, primer length and design,primer and dNTP concentrations, and buffer composition, to name but afew. Contamination of the reaction with exogenous DNA (e.g., DNA spilledonto lab surfaces) or cross-contamination is also a major consideration.Reaction conditions must be carefully optimized for each differentprimer pair and target sequence, and the process can take days, even foran experienced investigator. The laboriousness of this process,including numerous technical considerations and other factors, presentsa significant drawback to using PCR in the clinical setting. Indeed, PCRhas yet to penetrate the clinical market in a significant way. The sameconcerns arise with LCR, as LCR must also be optimized to use differentoligonucleotide sequences for each target sequence. In addition, bothmethods require expensive equipment, capable of precise temperaturecycling.

Many applications of nucleic acid detection technologies, such as instudies of allelic variation, involve not only detection of a specificsequence in a complex background, but also the discrimination betweensequences with few, or single, nucleotide differences. One method forthe detection of allele-specific variants by PCR is based upon the factthat it is difficult for Taq polymerase to synthesize a DNA strand whenthere is a mismatch between the template strand and the 3′ end of theprimer. An allele-specific variant may be detected by the use of aprimer that is perfectly matched with only one of the possible alleles;the mismatch to the other allele acts to prevent the extension of theprimer, thereby preventing the amplification of that sequence. Thismethod has a substantial limitation in that the base composition of themismatch influences the ability to prevent extension across themismatch, and certain mismatches do not prevent extension or have only aminimal effect (Kwok et al., Nuel. Acids Res., 18:999 [1990]).)

A similar 3′-mismatch strategy is used with greater effect to preventligation in the LCR (Barany, PCR Meth. Applic., 1:5 [1991]). Anymismatch effectively blocks the action of the thermostable ligase, butLCR still has the drawback of target-independent background ligationproducts initiating the amplification. Moreover, the combination of PCRwith subsequent LCR to identify the nucleotides at individual positionsis also a clearly cumbersome proposition for the clinical laboratory.

II. Direct Detection Technology

When a sufficient amount of a nucleic acid to be detected is available,there are advantages to detecting that sequence directly, instead ofmaking more copies of that target, (e.g., as in PCR and LCR). Mostnotably, a method that does not amplify the signal exponentially is moreamenable to quantitative analysis. Even if the signal is enhanced byattaching multiple dyes to a single oligonucleotide, the correlationbetween the final signal intensity and amount of target is direct. Sucha system has an additional advantage that the products of the reactionwill not themselves promote further reaction, so contamination of labsurfaces by the products is not as much of a concern. Traditionalmethods of direct detection including Northern and Southern blotting andRNase protection assays usually require the use of radioactivity and arenot amenable to automation. Recently devised techniques have sought toeliminate the use of radioactivity and/or improve the sensitivity inautomatable formats. Two examples are the “Cycling Probe Reaction”(CPR), and “Branched DNA” (bDNA)

The cycling probe reaction (CPR) (Duck et al., BioTech., 9:142 [1990]),uses a long chimeric oligonucleotide in which a central portion is madeof RNA while the two termini are made of DNA. Hybridization of the probeto a target DNA and exposure to a thermostable RNase H causes the RNAportion to be digested. This destabilizes the remaining DNA portions ofthe duplex, releasing the remainder of the probe from the target DNA andallowing another probe molecule to repeat the process. The signal, inthe form of cleaved probe molecules, accumulates at a linear rate. Whilethe repeating process increases the signal, the RNA portion of theoligonucleotide is vulnerable to RNases that may be carried throughsample preparation.

Branched DNA (bDNA), described by Urdea et al., Gene 61:253-264 (1987),involves oligonucleotides with branched structures that allow eachindividual oligonucleotide to carry 35 to 40 labels (e.g., alkalinephosphatase enzymes). While this enhances the signal from ahybridization event, signal from non-specific binding is similarlyincreased.

While both of these methods have the advantages of direct detectiondiscussed above, neither the CPR or bDNA methods can make use of thespecificity allowed by the requirement of independent recognition by twoor more probe (oligonucleotide) sequences, as is common in the signalamplification methods described in section I. above. The requirementthat two oligonucleotides must hybridize to a target nucleic acid inorder for a detectable signal to be generated confers an extra measureof stringency on any detection assay. Requiring two oligonucleotides tobind to a target nucleic acid reduces the chance that false “positive”results will be produced due to the non-specific binding of a probe tothe target. The further requirement that the two oligonucleotides mustbind in a specific orientation relative to the target,as is required inPCR, where oligonucleotides must be oppositely but appropriatelyoriented such that the DNA polymerase can bridge the gap between the twooligonucleotides in both directions, further enhances specificity of thedetection reaction. However, it is well known to those in the art thateven though PCR utilizes two oligonucleotide probes (termed primers)“non-specific” amplification (i.e., amplification of sequences notdirected by the two primers used) is a common artifact. This is in partbecause the DNA polymerase used in PCR can accommodate very largedistances, measured in nucleotides, between the oligonucleotides andthus there is a large window in which non-specific binding of anoligonucleotide can lead to exponential amplification of inappropriateproduct. The LCR, in contrast, cannot proceed unless theoligonucleotides used are bound to the target adjacent to each other andso the full benefit of the dual oligonucleotide hybridization isrealized.

An ideal direct detection method would combine the advantages of thedirect detection assays (e.g., easy quantification and minimal risk ofcarry-over contamination) with the specificity provided by a dualoligonucleotide hybridization assay.

SUMMARY OF THE INVENTION

The present invention relates to means for cleaving a nucleic acidcleavage structure in a site-specific manner. In one embodiment, themeans for cleaving is a cleaving enzyme comprising 5′ nucleases derivedfrom thermostable DNA polymerases. These polymerases form the basis of anovel method of detection of specific nucleic acid sequences. Thepresent invention contemplates use of novel detection methods forvarious uses, including, but not limited to clinical diagnosticpurposes.

In one embodiment, the present invention contemplates a DNA sequenceencoding a DNA polymerase altered in sequence (i.e., a “mutant” DNApolymerase) relative to the native sequence, such that it exhibitsaltered DNA synthetic activity from that of the native (i.e., “wildtype”) DNA polymerase. It is preferred that the encoded DNA polymeraseis altered such that it exhibits reduced synthetic activity compared tothat of the native DNA polymerase. In this manner, the enzymes of theinvention are predominantly 5′ nucleases and are capable of cleavingnucleic acids in a structure-specific manner in the absence ofinterfering synthetic activity.

Importantly, the 5′ nucleases of the present invention are capable ofcleaving linear duplex structures to create single discrete cleavageproducts. These linear structures are either 1) not cleaved by the wildtype enzymes (to any significant degree), or 2) are cleaved by the wildtype enzymes so as to create multiple products. This characteristic ofthe 5′ nucleases has been found to be a consistent property of enzymesderived in this manner from thermostable polymerases across eubacterialthermophilic species.

It is not intended that the invention be limited by the nature of thealteration necessary to render the polymerase synthesis-deficient. Noris it intended that the invention be limited by the extent of thedeficiency. The present invention contemplates various structures,including altered structures (primary, secondary, etc.), as well asnative structures, that may be inhibited by synthesis inhibitors.

Where the polymerase structure is altered, it is not intended that theinvention be limited by the means by which the structure is altered. Inone embodiment, the alteration of the native DNA sequence comprises achange in a single nucleotide. In another embodiment, the alteration ofthe native DNA sequence comprises a deletion of one or more nucleotides.In yet another embodiment, the alteration of the native DNA sequencecomprises an insertion of one or more nucleotides. It is contemplatedthat the change in DNA sequence may manifest itself as change in aminoacid sequence.

The present invention contemplates structure-specific nucleases from avariety of sources, including mesophilic, psychrophilic, thermophilic,and hyperthermophilic organisms. The preferred structure-specificnucleases are thermostable. Thermostable structure-specific nucleasesare contemplated as particularly useful in that they operate attemperatures where nucleic acid hybridization is extremely specific,allowing for allele-specific detection (including single-basemismatches). In one embodiment, the thermostable structure-specific arethermostable 5′ nucleases which are selected from the group consistingof altered polymerases derived from the native polymerases of Thermusspecies, including, but not limited to Thermus aquaticus, Thermusflavus, and Thermus thermophilus. However, the invention is not limitedto the use of thermostable 5′ nucleases. Thermostable structure-specificnucleases from the FEN-1, RAD2 and XPG class of nucleases are alsopreferred.

The present invention provides a composition comprising a cleavagestructure, said cleavage structure comprising: a) a target nucleic acid,said target nucleic acid having a first region, a second region, a thirdregion and a fourth region, wherein said first region is locatedadjacent to and downstream from said second region, said second regionis located adjacent to and downstream from said third region and saidthird region is located adjacent to and downstream from said fourthregion; b) a first oligonucleotide complementary to said fourth regionof said target nucleic acid; c) a second oligonucleotide having a 5′portion and a 3′ portion wherein said 5′ portion of said secondoligonucleotide contains a sequence complementary to said second regionof said target nucleic acid and wherein said 3′ portion of said secondoligonucleotide contains a sequence complementary to said third regionof said target nucleic acid; and d) a third oligonucleotide having a 5′portion and a 3′ portion wherein said 5′ portion of said thirdoligonucleotide contains a sequence complementary to said first regionof said target nucleic acid and wherein said 3′ portion of said thirdoligonucleotide contains a sequence complementary to said second regionof said target nucleic acid.

The present invention is not limited by the length of the four regionsof the target nucleic acid. In one embodiment, the first region of thetarget nucleic acid has a length of 11 to 50 nucleotides. In anotherembodiment, the second region of the target nucleic acid has a length ofone to three nucleotides. In another embodiment, the third region of thetarget nucleic acid has a length of six to nine nucleotides. In yetanother embodiment, the fourth region of the target nucleic acid has alength of 6 to 50 nucleotides.

The invention is not limited by the nature or composition of the of thefirst, second, third and fourth oligonucleotides; these oligonucleotidesmay comprise DNA, RNA, PNA and combinations thereof as well as comprisemodified nucleotides, universal bases, adducts, etc. Further, one ormore of the first, second, third and the fourth oligonucleotides maycontain a dideoxynucleotide at the 3′ terminus.

In a preferred embodiment, the target nucleic acid is not completelycomplementary to at least one of the first, the second, the third andthe fourth oligonucleotides. In a particularly preferred embodiment, thetarget nucleic acid is not completely complementary to the secondoligonucleotide.

As noted above, the present invention contemplates the use ofstructure-specific nucleases in a detection method. In one embodiment,the present invention provides a method of of detecting the presence ofa target nucleic acid molecule by detecting non-target cleavage productscomprising: a) providing: i) a cleavage means, ii) a source of targetnucleic acid, the target nucleic acid having a first region, a secondregion, a third region and a fourth region, wherein the first region islocated adjacent to and downstream from the second region, the secondregion is located adjacent to and downstream from the third region andthe third region is located adjacent to and downstream from the fourthregion; iii) a first oligonucleotide complementary to the fourth regionof the target nucleic acid; iv) a second oligonucleotide having a 5′portion and a 3′ portion wherein the 5′ portion of the secondoligonucleotide contains a sequence complementary to the second regionof said target nucleic acid and wherein the 3′ portion of the secondoligonucleotide contains a sequence complementary to the third region ofthe target nucleic acid; iv) a third oligonucleotide having a 5′ and a3′ portion wherein the 5′ portion of the third oligonucleotide containsa sequence complementary to the first region of the target nucleic acidand wherein the 3′ portion of the third oligonucleotide contains asequence complementary to the second region of the target nucleic acid;b) mixing the cleavage means, the target nucleic acid, the firstoligonucleotide, the second oligonucleotide and the thirdoligonucleotide to create a reaction mixture under reaction conditionssuch that the first oligonucleotide is annealed to the fourth region ofthe target nucleic acid and wherein at least the 3′ portion of thesecond oligonucleotide is annealed to the target nucleic acid andwherein at least the 5′ portion of the third oligonucleotide is annealedto the target nucleic acid so as to create a cleavage structure andwherein cleavage of the cleavage structure occurs to generate non-targetcleavage products, each non-target cleavage product having a 3′-hydroxylgroup; and c) detecting the non-target cleavage products.

The invention is not limited by the nature of the target nucleic acid.In one embodiment, the target nucleic acid comprises single-strandedDNA. In another embodiment, the target nucleic acid comprisesdouble-stranded DNA and prior to step c), the reaction mixture istreated such that the double-stranded DNA is rendered substantiallysingle-stranded. In another embodiment, the target nucleic acidcomprises RNA and the first and second oligonucleotides comprise DNA.

The invention is not limited by the nature of the cleavage means. In oneembodiment, the cleavage means is a structure-specific nuclease;particularly preferred structure-specific nucleases are thermostablestructure-specific nucleases. In a preferrred embodiment, thethermostable structure-specific nuclease is encoded by a DNA sequenceselected from the group consisting of SEQ ID NOS:1-3, 9, 10, 12, 21, 30,and 31.

In a preferred embodiment, the detection of the non-target cleavageproducts comprises electrophoretic separation of the products of thereaction followed by visualization of the separated non-target cleavageproducts.

In another preferred embodiment, one or more of the first, second, andthird oligonucleotides contain a dideoxynucleotide at the 3′ terminus.When dideoxynucleotide-containing oligonucleotides are employed, thedetection of the non-target cleavage products preferably comprises: a)incubating said non-target cleavage products with a template-independentpolymerase and at least one labelled nucleoside triphosphate underconditions such that at least one labelled nucleotide is added to the3′-hydroxyl group of said non-target cleavage products to generatelabelled non-target cleavage products; and b) detecting the presence ofsaid labelled non-target cleavage products. The invention is not limitedby the nature of the template-independent polymerase employed; in oneembodiment, the template-independent polymerase is selected from thegroup consisting of terminal deoxynucleotidyl transferase (TdT) and polyA polymerase. When TdT or polyA polymerase are employed in the detectionstep, the second oligonucleotide may contain a 5′ end label, the 5′ endlabel being a different label than the label present upon the labellednucleoside triphosphate. The invention is not limited by the nature ofthe 5′ end label; a wide variety of suitable 5′ end labels are known tothe art and include biotin, fluorescein, tetrachlorofluorescein,hexachlorofluorescein, Cy3 amidite, Cy5 amidite and digoxigenin.

In another embodiment, detecting the non-target cleavage productscomprises: a) incubating said non-target cleavage products with atemplate-independent polymerase and at least one nucleoside triphosphateunder conditions such that at least one nucleotide is added to the3′-hydroxyl group of the non-target cleavage products to generate tailednon-target cleavage products; and b) detecting the presence of thetailed non-target cleavage products. The invention is not limited by thenature of the template-independent polymerase employed; in oneembodiment, the template-independent polymerase is selected from thegroup consisting of terminal deoxynucleotidyl transferase (TdT) and polyA polymerase. When TdT or polyA polymerase are employed in the detectionstep, the second oligonucleotide may contain a 5′ end label. Theinvention is not limited by the nature of the 5′ end label; a widevariety of suitable 5′ end labels are known to the art and includebiotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3amidite, Cy5 amidite and digoxigenin.

In a preferred embodiment, the reaction conditions comprise providing asource of divalent cations; particularly preferred divalent cations areMn²⁺ and Mg²⁺ ions.

The present invention further provides a method of detecting thepresence of a target nucleic acid molecule by detecting non-targetcleavage products comprising: a) providing: i) a cleavage means, ii) asource of target nucleic acid, said target nucleic acid having a firstregion, a second region and a third region, wherein said first region islocated adjacent to and downstream from said second region and whereinsaid second region is located adjacent to and downstream from said thirdregion; iii) a first oligonucleotide having a 5′ and a 3′ portionwherein said 5′ portion of said first oligonucleotide contains asequence complementary to said second region of said target nucleic acidand wherein said 3′ portion of said first oligonucleotide contains asequence complementary to said third region of said target nucleic acid;iv) a second oligonucleotide having a length between eleven to fifteennucleotides and further having a 5′ and a 3′ portion wherein said 5′portion of said second oligonucleotide contains a sequence complementaryto said first region of said target nucleic acid and wherein said 3′portion of said second oligonucleotide contains a sequence complementaryto said second region of said target nucleic acid; b) mixing saidcleavage means, said target nucleic acid, said first oligonucleotide andsaid second oligonucleotide to create a reaction mixture under reactionconditions such that at least said 3′ portion of said firstoligonucleotide is annealed to said target nucleic acid and wherein atleast said 5′ portion of said second oligonucleotide is annealed to saidtarget nucleic acid so as to create a cleavage structure and whereincleavage of said cleavage structure occurs to generate non-targetcleavage products, each non-target cleavage product having a 3′-hydroxylgroup; and c) detecting said non-target cleavage products. In apreferred embodiment the cleavage means is a structure-specificnuclease, preferably a thermostable structure-specific nuclease.

The invention is not limited by the length of the various regions of thetarget nucleic acid. In a preferred embodiment, the second region ofsaid target nucleic acid has a length between one to five nucleotides.In another preferred embodiment, one or more of the first and the secondoligonucleotides contain a dideoxynucleotide at the 3′ terminus. Whendideoxynucleotide-containing oligonucleotides are employed, thedetection of the non-target cleavage products preferably comprises: a)incubating said non-target cleavage products with a template-independentpolymerase and at least one labelled nucleoside triphosphate underconditions such that at least one labelled nucleotide is added to the3′-hydroxyl group of said non-target cleavage products to generatelabelled non-target cleavage products; and b) detecting the presence ofsaid labelled non-target cleavage products. The invention is not limitedby the nature of the template-independent polymerase employed; in oneembodiment, the template-independent polymerase is selected from thegroup consisting of terminal deoxynucleotidyl transferase (TdT) and polyA polymerase. When TdT or polyA polymerase are employed in the detectionstep, the second oligonucleotide may contain a 5′ end label, the 5′ endlabel being a different label than the label present upon the labellednucleoside triphosphate. The invention is not limited by the nature ofthe 5′ end label; a wide variety of suitable 5′ end labels are known tothe art and include biotin, fluorescein, tetrachlorofluorescein,hexachlorofluorescein, Cy3 amidite, Cy5 amidite and digoxigenin.

In another embodiment, detecting the non-target cleavage productscomprises: a) incubating said non-target cleavage products with atemplate-independent polymerase and at least one nucleoside triphosphateunder conditions such that at least one nucleotide is added to the3′-hydroxyl group of the non-target cleavage products to generate tailednon-target cleavage products; and b) detecting the presence of thetailed non-target cleavage products. The invention is not limited by thenature of the template-independent polymerase employed; in oneembodiment, the template-independent polymerase is selected from thegroup consisting of terminal deoxynucleotidyl transferase (TdT) and polyA polymerase. When TdT or polyA polymerase are employed in the detectionstep, the second oligonucleotide may contain a 5′ end label. Theinvention is not limited by the nature of the 5′ end label; a widevariety of suitable 5′ end labels are known to the art and includebiotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3amidite, Cy5 amidite and digoxigenin.

The novel detection methods of the invention may be employed for thedetection of target DNAs and RNAs including, but not limited to, targetDNAs and RNAs comprising wild type and mutant alleles of genes,including genes from humans or other animals that are or may beassociated with disease or cancer. In addition, the methods of theinvention may be used for the detection of and/or identification ofstrains of microorganisms, including bacteria, fungi, protozoa, ciliatesand viruses (and in particular for the detection and identification ofRNA viruses, such as HCV).

DESCRIPTION OF THE DRAWINGS

FIG. 1A provides a schematic of one embodiment of the detection methodof the present invention.

FIG. 1B provides a schematic of a second embodiment of the detectionmethod of the present invention.

FIG. 2 is a comparison of the nucleotide structure of the DNAP genesisolated from Thermus aquaticus (SEQ ID NO:1), Thermus flavus (SEQ IDNO:2) and Thermus thermophilus (SEQ ED NO:3); the consensus sequence(SEQ ID NO:7) is shown at the top of each row.

FIG. 3 is a comparison of the amino acid sequence of the DNAP isolatedfrom Thermus aquaticus (SEQ ID NO:4), Thermus flavus (SEQ ID NO:5), andThermus thermophilus (SEQ ID NO:6); the consensus sequence (SEQ ID NO:8)is shown at the top of each row.

FIGS. 4A-G are a set of diagrams of wild-type and synthesis-deficientDNAPTaq genes.

FIG. 5A depicts the wild-type Thermus flavus polymerase gene.

FIG. 5B depicts a synthesis-deficient Thermus flavus polymerase gene.

FIG. 6 depicts a structure which cannot be amplified using DNAPTaq.

FIG. 7 is a ethidium bromide-stained gel demonstrating attempts toamplify a bifurcated duplex using either DNAPTaq or DNAPStf (i.e., theStoffel fragment of DNAPTaq).

FIG. 8 is an autoradiogram of a gel analyzing the cleavage of abifurcated duplex by DNAPTaq and lack of cleavage by DNAPStf.

FIGS. 9A-B are a set of autoradiograms of gels analyzing cleavage orlack of cleavage upon addition of different reaction components andchange of incubation temperature during attempts to cleave a bifurcatedduplex with DNAPTaq.

FIGS. 10A-B are an autoradiogram displaying timed cleavage reactions,with and without primer.

FIGS. 11A-B are a set of autoradiograms of gels demonstrating attemptsto cleave a bifurcated duplex (with and without primer) with variousDNAPs.

FIGS. 12A shows the substrates and oligonucleotides used to test thespecific cleavage of substrate DNAs targeted by pilot oligonucleotides.

FIG. 12B shows an autoradiogram of a gel showing the results of cleavagereactions using the substrates and oligonucleotides shown FIG. 12A.

FIG. 13A shows the substrate and oligonucleotide used to test thespecific cleavage of a substrate RNA targeted by a pilotoligonucleotide.

FIG. 13B shows an autoradiogram of a gel showing the results of acleavage reaction using the substrate and oligonucleotide shown in FIG.13A.

FIG. 14 is a diagram of vector pTTQ18.

FIG. 15 is a diagram of vector pET-3c.

FIG. 16A-E depicts a set of molecules which are suitable substrates forcleavage by the 5′ nuclease activity of DNAPs.

FIG. 17 is an autoradiogram of a gel showing the results of a cleavagereaction run with synthesis-deficient DNAPs.

FIG. 18 is an autoradiogram of a PEI chromatogram resolving the productsof an assay for synthetic activity in synthesis-deficient DNAPTaqclones.

FIG. 19A depicts the substrate molecule used to test the ability ofsynthesis-deficient DNAPs to cleave short hairpin structures.

FIG. 19B shows an autoradiogram of a gel resolving the products of acleavage reaction run using the substrate shown in FIG. 19A.

FIG. 20A shows the A- and T-hairpin molecules used in thetrigger/detection assay.

FIG. 20B shows the sequence of the alpha primer used in thetrigger/detection assay.

FIG. 20C shows the structure of the cleaved A- and T-hairpin molecules.

FIG. 20D depicts the complementarity between the A- and T-hairpinmolecules.

FIG. 21 provides the complete 206-mer duplex sequence employed as asubstrate for the 5′ nucleases of the present invention

FIGS. 22A and B show the cleavage of linear nucleic acid substrates(based on the 206-mer of FIG. 21) by wild type DNAPs and 5′ nucleasesisolated from Thermus aquaticus and Thermus flavus.

FIG. 23 provides a detailed schematic corresponding to the of oneembodiment of the detection method of the present invention.

FIG. 24 shows the propagation of cleavage of the linear duplex nucleicacid structures of FIG. 23 by the 5′ nucleases of the present invention.

FIG. 25A shows the “nibbling” phenomenon detected with the DNAPs of thepresent invention.

FIG. 25B shows that the “nibbling” of FIG. 25A is 5′ nucleolyticcleavage and not phosphatase cleavage.

FIG. 26 demonstrates that the “nibbling” phenomenon is duplex dependent.

FIG. 27 is a schematic showing how “nibbling” can be employed in adetection assay.

FIG. 28 demonstrates that “nibbling” can be target directed.

FIG. 29 provides a schematic drawing of a target nucleic acid with aninvader oligonucleotide and a probe oligonucleotide annealed to thetarget.

FIG. 30 provides a schematic showing the S-60 hairpin oligonucleotide(SEQ ID NO:40) with the annealed P-15 oligonucletide (SEQ ID NO:41).

FIG. 31 is an autoradiogram of a gel showing the results of a cleavagereaction run using the S-60 hairpin in the presence or absence of theP-15 oligonucleotide.

FIG. 32 provides a schematic showing three different arrangements oftarget-specific oligonucleotides and their hybridization to a targetnucleic acid which also has a probe oligonucleotide annealed thereto.

FIG. 33 is the image generated by a fluorescence imager showing that thepresence of an invader oligonucleotide causes a shift in the site ofcleavage in a probe/target duplex.

FIG. 34 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run using the threetarget-specific oligonucleotides diagrammed in FIG. 32.

FIG. 35 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run in the presence orabsence of non-target nucleic acid molecules.

FIG. 36 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run in the presence ofdecreasing amounts of target nucleic acid.

FIG. 37 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run in the presence orabsence of saliva extract using various thermostable 5′ nucleases or DNApolymerases.

FIG. 38 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run using various 5′nucleases.

FIG. 39 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run using two targetnucleic acids which differ by a single basepair at two differentreaction temperatures.

FIG. 40A provides a schematic showing the effect of elevated temperatureupon the annealing and cleavage of a probe oligonucleotide along atarget nucleic acid wherein the probe contains a region ofnoncomplementarity with the target.

FIG. 40B provides a schematic showing the effect of adding an upstreamoligonucleotide upon the annealing and cleavage of a probeoligonucleotide along a target nucleic acid wherein the probe contains aregion of noncomplementarity with the target.

FIG. 41 provides a schematic showing an arrangement of a target-specificinvader oligonucleotide (SEQ ID NO:50) and a target-specific probeoligonucleotide (SEQ ID NO:49) bearing a 5′ Cy3 label along a targetnucleic acid (SEQ ID NO:42).

FIG. 42 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run in the presence ofincreasing concentrations of KCl.

FIG. 43 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run in the presence ofincreasing concentrations of NaCl.

FIG. 44 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run in the presence ofincreasing concentrations of LiCl.

FIG. 45 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run in the presence ofincreasing concentrations of KGlu.

FIG. 46 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run in the presence ofincreasing concentrations of MnCl₂ or MgCl₂.

FIG. 47 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run in the presence ofincreasing concentrations of CTAB.

FIG. 48 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run in the presence ofincreasing concentrations of PEG.

FIG. 49 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run in the presence ofglycerol, Tween-20 and/or Nonidet-P40.

FIG. 50 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run in the presence ofincreasing concentrations of gelatin in reactions containing or lackingKCl or LiCl.

FIG. 51 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run in the presence ofincreasing amounts of genomic DNA or tRNA.

FIG. 52 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run use a HCV RNA target.

FIG. 53 is the image generated by a fluorescence imager showing theproducts of invader-directed cleavage assays run using a HCV RNA targetand demonstrate the stability of RNA targets under invader-directedcleavage assay conditions.

FIG. 54 is the image generated by a fluorescence imager showing thesensitivity of detection and the stability of RNA in invader-directedcleavage assays run using a HCV RNA target.

FIG. 55 is the image generated by a fluorescence imager showing thermaldegradation of oligonucleotides containing or lacking a 3′ phosphategroup.

FIG. 56 depicts the structure of amino-modified oligonucleotides 70 and74.

FIG. 57 depicts the structure of amino-modified oligonucleotide 75

FIG. 58 depicts the structure of amino-modified oligonucleotide 76.

FIG. 59 is the image generated by a fluorescence imager scan of an IEFgel showing the migration of substrates 70, 70 dp, 74, 74 dp, 75, 75 dp,76 and 76 dp.

FIG. 60A provides a schematic showing an arrangement of atarget-specific invader oligonucleotide (SEQ ID NO:61) and atarget-specific probe oligonucleotide (SEQ ID NO:62) bearing a 5′ Cy3label along a target nucleic acid (SEQ ID NO:63).

FIG. 60B is the image generated by a fluorescence imager showing thedetection of specific cleavage products generated in an invasivecleavage assay using charge reversal (i.e., charge based separation ofcleavage products).

FIG. 61 is the image generated by a fluorescence imager which depictsthe sensitivity of detection of specific cleavage products generated inan invasive cleavage assay using charge reversal.

FIG. 62 depicts a first embodiment of a device for the charge-basedseparation of oligonucleotides.

FIG. 63 depicts a second embodiment of a device for the charge-basedseparation of oligonucleotides.

FIG. 64 shows an autoradiogram of a gel showing the results of cleavagereactions run in the presence or absence of a primer oligonucleotide; asequencing ladder is shown as a size marker.

FIGS. 65 a-d depict four pairs of oligonucleotides; in each pair shown,the upper arrangement of a probe annealed to a target nucleic acid lacksan upstream oligonucleotide and the lower arrangement contains anupstream oligonucleotide.

FIG. 66 shows the chemical structure of several positively chargedheterodimeric DNA-binding dyes.

FIG. 67 is a schematic showing alternative methods for the tailing anddetection of specific cleavage products in the context of theInvader™-directed cleavage assay.

FIG. 68 provides a schematic drawing of a target nucleic acid with anInvader™ oligonucleotide, a miniprobe, and a stacker oligonucleotideannealed to the target.

FIG. 69 provides a space-filling model of the 3-dimensional structure ofthe T5 5′-exonuclease.

FIG. 70 provides an alignment of the amino acid sequences of severalFEN-1 nucleases including the Methanococcus jannaschii FEN-1 protein(MJAFEN1.PRO), the Pyrococcus furiosus FEN-1 protein (PFUFEN1.PRO), thehuman FEN-1 protein (HUMFEN1.PRO), the mouse FEN-1 protein(MUSFEN1.PRO), the Saccharomyces cerevisiae YKL510 protein (YST510.PRO),the Saccharomyces cerevisiae RAD2 protein (YSTRAD2.PRO), theShizosaccharomyces pombe RAD13 protein (SPORAD13.PRO), the human XPGprotein (HUMXPG.PRO), the mouse XPG protein (MUSXPG.PRO), the Xenopuslaevis XPG protein (XENXPG.PRO) and the C. elegans RAD2 protein(CELRAD2.PRO); portions of the amino acid sequence of some of theseproteins were not shown in order to maximize the alignment betweenproteins. The numbers to the left of each line of sequence refers to theamino acid residue number; dashes represent gaps introduced to maximizealignment.

FIG. 71 provides a schematic showing the S-33 and 11-8-0oligonucleotides in a folded configuration; the cleavage site isindicated by the arrowhead.

DEFINITIONS

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides such asan oligonucleotide or a target nucleic acid) related by the base-pairingrules. For example, for the sequence “A-G-T,” is complementary to thesequence “T-C-A.” Complementarity may be “partial,” in which only someof the nucleic acids' bases are matched according to the base pairingrules. Or, there may be “complete” or “total” complementarity betweenthe nucleic acids. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methodswhich depend upon binding between nucleic acids.

The term “homology” refers to a degree of identity. There may be partialhomology or complete homology. A partially identical sequence is onethat is less than 100% identical to another sequence.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (see e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization (1985). Other referencesinclude more sophisticated computations which take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds, under which nucleic acid hybridizations are conducted. With“high stringency” conditions, nucleic acid base pairing will occur onlybetween nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “weak” or “low”stringency are often required when it is desired that nucleic acidswhich are not completely complementary to one another be hybridized orannealed together.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor. The polypeptide can be encoded by a full length codingsequence or by any portion of the coding sequence so long as the desiredenzymatic activity is retained.

The term “wild-type” refers to a gene or gene product which has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product which displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally-occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product.

The term “recombinant DNA vector” as used herein refers to DNA sequencescontaining a desired coding sequence and appropriate DNA sequencesnecessary for the expression of the operably linked coding sequence in aparticular host organism. DNA sequences necessary for expression inprocaryotes include a promoter, optionally an operator sequence, aribosome binding site and possibly other sequences. Eukaryotic cells areknown to utilize promoters, polyadenlyation signals and enhancers.

The term “LTR” as used herein refers to the long terminal repeat foundat each end of a provirus (Le., the integrated form of a retrovirus).The LTR contains numerous regulatory signals including transcriptionalcontrol elements, polyadenylation signals and sequences needed forreplication and integration of the viral genome. The viral LTR isdivided into three regions called U3, R and U5.

The U3 region contains the enhancer and promoter elements. The U5 regioncontains the polyadenylation signals. The R (repeat) region separatesthe U3 and U5 regions and transcribed sequences of the R region appearat both the 5′ and 3′ ends of the viral RNA.

The term “oligonucleotide” as used herein is defined as a moleculecomprised of two or more deoxyribonucleotides or ribonucleotides,preferably at least 5 nucleotides, more preferably at least about 10-15nucleotides and more preferably at least about 15 to 30 nucleotides. Theexact size will depend on many factors, which in turn depends on theultimate function or use of the oligonucleotide. The oligonucleotide maybe generated in any manner, including chemical synthesis, DNAreplication, reverse transcription, or a combination thereof.

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. A first regionalong a nucleic acid strand is said to be upstream of another region ifthe 3′ end of the first region is before the 5′ end of the second regionwhen moving along a strand of nucleic acid in a 5′ to 3′ direction.

When two different, non-overlapping oligonucleotides anneal to differentregions of the same linear complementary nucleic acid sequence, and the3′ end of one oligonucleotide points towards the 5′ end of the other,the former may be called the “upstream” oligonucleotide and the latterthe “downstream” oligonucleotide.

The term “primer” refers to an oligonucleotide which is capable ofacting as a point of initiation of synthesis when placed underconditions in which primer extension is initiated. An oligonucleotide“primer” may occur naturally, as in a purified restriction digest or maybe produced synthetically.

A primer is selected to be “substantially” complementary to a strand ofspecific sequence of the template. A primer must be sufficientlycomplementary to hybridize with a template strand for primer elongationto occur. A primer sequence need not reflect the exact sequence of thetemplate. For example, a non-complementary nucleotide fragment may beattached to the 5′ end of the primer, with the remainder of the primersequence being substantially complementary to the strand.Non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence has sufficient complementaritywith the sequence of the template to hybridize and thereby form atemplate primer complex for synthesis of the extension product of theprimer.

“Hybridization” methods involve the annealing of a complementarysequence to the target nucleic acid (the sequence to be detected; thedetection of this sequence may be by either direct or indirect means).The ability of two polymers of nucleic acid containing complementarysequences to find each other and anneal through base pairing interactionis a well-recognized phenomenon. The initial observations of the“hybridization” process by Marmur and Lane, Proc. NatL Acad. Sci. USA46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461 (1960)have been followed by the refinement of this process into an essentialtool of modern biology.

With regard to complementarity, it is important for some diagnosticapplications to determine whether the hybridization represents completeor partial complementarity. For example, where it is desired to detectsimply the presence or absence of pathogen DNA (such as from a virus,bacterium, fungi, mycoplasma, protozoan) it is only important that thehybridization method ensures hybridization when the relevant sequence ispresent; conditions can be selected where both partially complementaryprobes and completely complementary probes will hybridize. Otherdiagnostic applications, however, may require that the hybridizationmethod distinguish between partial and complete complementarity. It maybe of interest to detect genetic polymorphisms. For example, humanhemoglobin is composed, in part, of four polypeptide chains. Two ofthese chains are identical chains of 141 amino acids (alpha chains) andtwo of these chains are identical chains of 146 amino acids (betachains). The gene encoding the beta chain is known to exhibitpolymorphism. The normal allele encodes a beta chain having glutamicacid at the sixth position. The mutant allele encodes a beta chainhaving valine at the sixth position. This difference in amino acids hasa profound (most profound when the individual is homozygous for themutant allele) physiological impact known clinically as sickle cellanemia. It is well known that the genetic basis of the amino acid changeinvolves a single base difference between the normal allele DNA sequenceand the mutant allele DNA sequence.

The complement of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.” Certain bases not commonly found innatural nucleic acids may be included in the nucleic acids of thepresent invention and include, for example, inosine and 7-deazaguanine.Complementarity need not be perfect; stable duplexes may containmismatched base pairs or unmatched bases. Those skilled in the art ofnucleic acid technology can determine duplex stability empiricallyconsidering a number of variables including, for example, the length ofthe oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

Stability of a nucleic acid duplex is measured by the meltingtemperature, or “T_(m).” The T_(m) of a particular nucleic acid duplexunder specified conditions is the temperature at which on average halfof the base pairs have disassociated.

The term “label” as used herein refers to any atom or molecule which canbe used to provide a detectable (preferably quantifiable) signal, andwhich can be attached to a nucleic acid or protein. Labels may providesignals detectable by fluorescence, radioactivity, colorimetry,gravimetry, X-ray diffraction or absorption, magnetism, enzymaticactivity, and the like. A label may be a charged moeity (positive ornegative charge) or alternatively, may be charge neutral.

The term “cleavage structure” as used herein, refers to a structurewhich is formed by the interaction of a probe oligonucleotide and atarget nucleic acid to form a duplex, said resulting structure beingcleavable by a cleavage means, including but not limited to an enzyme.The cleavage structure is a substrate for specific cleavage by saidcleavage means in contrast to a nucleic acid molecule which is asubstrate for non-specific cleavage by agents such as phosphodiesteraseswhich cleave nucleic acid molecules without regard to secondarystructure (i.e., no formation of a duplexed structure is required).

The term “cleavage means” as used herein refers to any means which iscapable of cleaving a cleavage structure, including but not limited toenzymes. The cleavage means may include native DNAPs having 5′ nucleaseactivity (e.g., Taq DNA polymerase, E. coli DNA polymerase I) and, morespecifically, modified DNAPs having 5′ nuclease but lacking syntheticactivity. The ability of 5′ nucleases to cleave naturally occurringstructures in nucleic acid templates (structure-specific cleavage) isuseful to detect internal sequence differences in nucleic acids withoutprior knowledge of the specific sequence of the nucleic acid. In thismanner, they are structure-specific enzymes. “Structure-specificnucleases” or “structure-specific enzymes” are enzymes which recognizespecific secondary structures in a nucleic molecule and cleave thesestructures. The cleavage means of the invention cleave a nucleic acidmolecule in response to the formation of cleavage structures; it is notnecessary that the cleavage means cleave the cleavage structure at anyparticular location within the cleavage structure.

The cleavage means is not restricted to enzymes having solely 5′nuclease activity. The cleavage means may include nuclease activityprovided from a variety of sources including the Cleavase® enzymes, theFEN-1 endonucleases (including RAD2 and XPG proteins), Taq DNApolymerase and E. coli DNA polymerase I.

The term “thermostable” when used in reference to an enzyme, such as a5′ nuclease, indicates that the enzyme is functional or active (i.e.,can perform catalysis) at an elevated temperature, i.e., at about 55° C.or higher.

The term “cleavage products” as used herein, refers to productsgenerated by the reaction of a cleavage means with a cleavage structure(i.e., the treatment of a cleavage structure with a cleavage means).

The term “target nucleic acid” refers to a nucleic acid molecule whichcontains a sequence which has at least partial complementarity with atleast a probe oligonucleotide and may also have at least partialcomplementarity with an invader oligonucleotide. The target nucleic acidmay comprise single- or double-stranded DNA or RNA.

The term “probe oligonucleotide” refers to an oligonucleotide whichinteracts with a target nucleic acid to form a cleavage structure in thepresence or absence of an invader oligonucleotide. When annealed to thetarget nucleic acid, the probe oligonucleotide and target form acleavage structure and cleavage occurs within the probe oligonucleotide.In the presence of an invader oligonucleotide upstream of the probeoligonucleotide along the target nucleic acid will shift the site ofcleavage within the probe oligonucleotide (relative to the site ofcleavage in the absence of the invader).

The term “non-target cleavage product” refers to a product of a cleavagereaction which is not derived from the target nucleic acid. As discussedabove, in the methods of the present invention, cleavage of the cleavagestructure occurs within the probe oligonucleotide. The fragments of theprobe oligonucleotide generated by this target nucleic acid-dependentcleavage are “non-target cleavage products.”

The term “invader oligonucleotide” refers to an oligonucleotide whichcontains sequences at its 3′ end which are substantially the same assequences located at the 5′ end of a probe oligonucleotide; theseregions will compete for hybridization to the same segment along acomplementary target nucleic acid.

The term “substantially single-stranded” when used in reference to anucleic acid substrate means that the substrate molecule existsprimarily as a single strand of nucleic acid in contrast to adouble-stranded substrate which exists as two strands of nucleic acidwhich are held together by inter-strand base pairing interactions.

The term “sequence variation” as used herein refers to differences innucleic acid sequence between two nucleic acids. For example, awild-type structural gene and a mutant form of this wild-type structuralgene may vary in sequence by the presence of single base substitutionsand/or deletions or insertions of one or more nucleotides. These twoforms of the structural gene are said to vary in sequence from oneanother. A second mutant form of the structural gene may exist. Thissecond mutant form is said to vary in sequence from both the wild-typegene and the first mutant form of the gene.

The term “liberating” as used herein refers to the release of a nucleicacid fragment from a larger nucleic acid fragment, such as anoligonucleotide, by the action of a 5′ nuclease such that the releasedfragment is no longer covalently attached to the remainder of theoligonucleotide.

The term “K_(m)” as used herein refers to the Michaelis-Menten constantfor an enzyme and is defined as the concentration of the specificsubstrate at which a given enzyme yields one-half its maximum velocityin an enzyme catalyzed reaction.

The term “nucleotide analog” as used herein refers to modified ornon-naturally occurring nucleotides such as 7-deaza purines (i.e.,7-deaza-dATP and 7-deaza-dGTP). Nucleotide analogs include base analogsand comprise modified forms of deoxyribonucleotides as well asribonucleotides.

The term “polymorphic locus” is a locus present in a population whichshows variation between members of the population (i.e., the most commonallele has a frequency of less than 0.95). In contrast, a “monomorphiclocus” is a genetic locus at little or no variations seen betweenmembers of the population (generally taken to be a locus at which themost common allele exceeds a frequency of 0.95 in the gene pool of thepopulation).

The term “microorganism” as used herein means an organism too small tobe observed with the unaided eye and includes, but is not limited tobacteria, virus, protozoans, fungi, and ciliates.

The term “microbial gene sequences” refers to gene sequences derivedfrom a microorganism.

The term “bacteria” refers to any bacterial species includingeubacterial and archaebacterial species.

The term “virus” refers to obligate, ultramicroscopic, intracellularparasites incapable of autonomous replication (i.e., replicationrequires the use of the host cell's machinery).

The term “multi-drug resistant” or multiple-drug resistant” refers to amicroorganism which is resistant to more than one of the antibiotics orantimicrobial agents used in the treatment of said microorganism.

The term “sample” in the present specification and claims is used in itsbroadest sense. On the one hand it is meant to include a specimen orculture (e.g., microbiological cultures). On the other hand, it is meantto include both biological and environmental samples.

Biological samples may be animal, including human, fluid, solid (e.g.stool) or tissue, as well as liquid and solid food and feed products andingredients such as dairy items, vegetables, meat and meat by-products,and waste. Biological samples may be obtained from all of the variousfamilies of domestic animals, as well as feral or wild animals,including, but not limited to, such animals as ungulates, bear, fish,lagamorphs, rodents, etc.

Environmental samples include environmental material such as surfacematter, soil, water and industrial samples, as well as samples obtainedfrom food and dairy processing instruments, apparatus, equipment,utensils, disposable and non-disposable items. These examples are not tobe construed as limiting the sample types applicable to the presentinvention.

The term “source of target nucleic acid” refers to any sample whichcontains nucleic acids (RNA or DNA). Particularly preferred sources oftarget nucleic acids are biological samples including, but not limitedto blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph,sputum and semen.

An oligonucleotide is said to be present in “excess” relative to anotheroligonucleotide (or target nucleic acid sequence) if thatoligonucleotide is present at a higher molar concentration that theother oligonucleotide (or target nucleic acid sequence). When anoligonucleotide such as a probe oligonucleotide is present in a cleavagereaction in excess relative to the concentration of the complementarytarget nucleic acid sequence, the reaction may be used to indicate theamount of the target nucleic acid present. Typically, when present inexcess, the probe oligonucleotide will be present at least a 100-foldmolar excess; typically at least 1 pmole of each probe oligonucleotidewould be used when the target nucleic acid sequence was present at about10 fmoles or less.

A sample “suspected of containing” a first and a second target nucleicacid may contain either, both or neither target nucleic acid molecule.

The term “charge-balanced” oligonucleotide refers to an olignucleotide(the input oligonucleotide in a reaction) which has been modified suchthat the modified oligonucleotide bears a charge, such that when themodified oligonucleotide is either cleaved (i.e., shortened) orelongated, a resulting product bears a charge different from the inputoligonucleotide (the “charge-unbalanced” oligonucleotide) therebypermitting separation of the input and reacted oligonucleotides on thebasis of charge. The term “charge-balanced” does not imply that themodified or balanced oligonucleotide has a net neutral charge (althoughthis can be the case). Charge-balancing refers to the design andmodification of an oligonucleotide such that a specific reaction productgenerated from this input oligonucleotide can be separated on the basisof charge from the input oligonuceotide.

For example, in an invader-directed cleavage assay in which the probeoligonucleotide bears the sequence: 5′-TTCTTTTCACCAGCGAGACGGG-3′ (i.e.,SEQ ID NO:61 without the modified bases) and cleavage of the probeoccurs between the second and third residues, one possiblecharge-balanced version of this oligonuceotide would be:5′-Cy3-AminoT-Amino-TCTTTTCACCAGCGAGAC GGG-3′. This modifiedoligonucleotide bears a net negative charge. After cleavage, thefollowing oligonucleotides are generated: 5′-Cy3-AminoT-Amino-T-3′ and5′-CTTTTCACCAGCGAGACGGG-3′ (residues 3-22of SEQ ID NO:61). 5′-Cy3-AminoT-Amino-T-3′ bears a detectable moeity (the positively-charged Cy3 dye)and two amino-modified bases. The amino-modified bases and the Cy3 dyecontribute positive charges in excess of the negative chargescontributed by the phosphate groups and thus the5′-Cy3-AminoT-Amino-T-3′oligonucleotide has a net positive charge. Theother, longer cleavage fragment, like the input probe, bears a netnegative charge. Because the 5′-Cy3-AminoT-Amino-T-3′fragment isseparable on the basis of charge from the input probe (thecharge-balanced oligonucleotide), it is referred to as acharge-unbalanced oligonucleotide. The longer cleavage product cannot beseparated on the basis of charge from the input oligonucleotide as botholigonucleotides bear a net negative charge; thus, the longer cleavageproduct is not a charge-unbalanced oligonucleotide.

The term “net neutral charge” when used in reference to anoligonucletide, including modified oligonucleotides, indicates that thesum of the charges present (i.e, R—NH³⁺ groups on thymidines, the N3nitrogen of cytosine, presence or absence or phosphate groups, etc.)under the desired reaction conditions is essentially zero. Anoligonucletide having a net neutral charge would not niigrate in anelectrical field.

The term “net positive charge” when used in reference to anoligonucletide, including modified oligonucleotides, indicates that thesum of the charges present (i.e, R—NH³⁺ groups on thymidines, the N3nitrogen of cytosine, presence or absence or phosphate groups, etc.)under the desired reaction conditions is +1 or greater. Anoligonucletide having a net positive charge would migrate toward thenegative electrode in an electrical field.

The term “net negative charge” when used in reference to anoligonucletide, including modified oligonucleotides, indicates that thesum of the charges present (i.e, R—NH³⁺ groups on thymidines, the N3nitrogen of cytosine, presence or absence or phosphate groups, etc.)under the desired reaction conditions is −1 or lower. An oligonucletidehaving a net negative charge would migrate toward the positive electrodein an electrical field.

The term “polymerization means” refers to any agent capable offacilitating the addition of nucleoside triphosphates to anoligonucleotide. Preferred polymerization means comprise DNApolymerases.

The term “ligation means” refers to any agent capable of facilitatig theligation (i.e., the formation of a phosphodiester bond between a 3′-OHand a 5′-P located at the termini of two strands of nuceic acid).Preferred ligation means comprise DNA ligases and RNA ligases.

The term “reactant” is used herein in its broadest sense. The reactantcan comprise an enzymatic reactant, a chemical reactant or ultravioletlight (ultraviolet light, particulary short wavelength ultraviolet lightis known to break oligonucleotide chains). Any agent capable of reactingwith an oligonucleotide to either shorten (i.e., cleave) or elongate theoligonucleotide is encompsased within the term “reactant.”

The term “adduct” is used herein in its broadest sense to indicate anycompound or element which can be added to an oligonucleotide. An adductmay be charged (postively or negatively) or may be charge neutral. Anadduct may be added to the oligonucleotide via covalent or non-covalentlinkages. Examples of adducts, include but are not limited toindodicarbocyanine dye amidites, amino-substituted nucleotides, ethidiumbromide, ethidium homodimer, (1,3-propanediamino)propidium,(diethylenetriamino)propidium, thiazole orange,(N-N′-tetramethyl-1,3-propanediamino)propyl thiazole orange,(N-N′-tetramethyl-1,2-ethanediamino)propyl thiazole orange, thiazoleorange-thiazole orange homodimer (TOTO), thiazole orande-thiazole blueheterodimer (TOTAB), thiazole orange-ethidium heterodimer 1 (TOED1),thiazole orange-ethidium heterodimer 2 (TOED2) and florescien-ethidiumheterodimer (FED), psoralens, biotin, streptavidin, avidin, etc.

Where a first oligonucleotide is complementary to a region of a targetnucleic acid and a second oligonucleotide has complementary to the sameregion (or a portion of this region) a “region of overlap” exists alongthe target nucleic acid. The degree of overlap will vary depending uponthe nature of the complementarity (see, e.g., region “X” in FIGS. 29 and67 and the accompanying discussions).

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample. For example, recombinant Cleavase®nucleases are expressed in bacterial host cells and the nucleases arepurified by the removal of host cell proteins; the percent of theserecombinant nucleases is thereby increased in the sample.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule which is comprised of segments of DNA joined together by meansof molecular biological techniques.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule which is expressed from arecombinant DNA molecule.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid.

“Nucleic acid sequence” as used herein refers to an oligonucleotide,nucleotide or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand. Similarly,“amino acid sequence” as used herein refers to peptide or proteinsequence.

“Peptide nucleic acid” (“PNA”) as used herein refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid [Nielsen PE et al. (1993)Anticancer Drug Des. 8:53-63].

As used herein, the term “substantially purified” refers to molecules,either nucleic or amino acid sequences, that are removed from theirnatural environment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated. An “isolated polynucleotide”or “isolated oligonucletide” is therefore a substantially purifiedpolynucleotide.

DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for treatingnucleic acid, and in particular, methods and compositions for detectionand characterization of nucleic acid sequences and sequence changes.

The present invention relates to means for cleaving a nucleic acidcleavage structure in a site-specific manner. In particular, the presentinvention relates to a cleaving enzyme having 5′ nuclease activitywithout interfering nucleic acid synthetic ability.

This invention provides 5′ nucleases derived from thermostable DNApolymerases which exhibit altered DNA synthetic activity from that ofnative thermostable DNA polymerases. The 5′ nuclease activity of thepolymerase is retained while the synthetic activity is reduced orabsent. Such 5′ nucleases are capable of catalyzing thestructure-specific cleavage of nucleic acids in the absence ofinterfering synthetic activity. The lack of synthetic activity during acleavage reaction results in nucleic acid cleavage products of uniformsize.

The novel properties of the nucleases of the invention form the basis ofa method of detecting specific nucleic acid sequences. This methodrelies upon the amplification of the detection molecule rather than uponthe amplification of the target sequence itself as do existing methodsof detecting specific target sequences.

DNA polymerases (DNAPs), such as those isolated from E. coli or fromthermophilic bacteria of the genus Thermus, are enzymes that synthesizenew DNA strands. Several of the known DNAPs contain associated nucleaseactivities in addition to the synthetic activity of the enzyme.

Some DNAPs are known to remove nucleotides from the 5′ and 3′ ends ofDNA chains [Komberg, DNA Replication, W. H. Freeman and Co., SanFrancisco, pp. 127-139 (1980)]. These nuclease activities are usuallyreferred to as 5′ exonuclease and 3′ exonuclease activities,respectively. For example, the 5′ exonuclease activity located in theN-terminal domain of several DNAPs participates in the removal of RNAprimers during lagging strand synthesis during DNA replication and theremoval of damaged nucleotides during repair. Some DNAPs, such as the E.coli DNA polymerase (DNAPEc1), also have a 3′ exonuclease activityresponsible for proof-reading during DNA synthesis (Komberg, supra).

A DNAP isolated from Thermus aquaticus, termed Taq DNA polymerase(DNAPTaq), has a 5′ exonuclease activity, but lacks a functional 3′exonucleolytic domain [Tindall and Kunkell, Biochem. 27:6008 (1988)].Derivatives of DNAPEc1 and DNAPTaq, respectively called the Klenow andStoffel fragments, lack 5′ exonuclease domains as a result of enzymaticor genetic manipulations [Brutlag et al., Biochem. Biophys. Res. Commun.37:982 (1969); Erlich et al., Science 252:1643 (1991); Setlow andKomberg, J. Biol. Chem. 247:232 (1972)].

The 5′ exonuclease activity of DNAPTaq was reported to requireconcurrent synthesis [Gelfand, PCR Technology—Principles andApplications for DNA Amplification (H. A. Erlich, Ed.), Stockton Press,New York, p. 19 (1989)]. Although mononucleotides predominate among thedigestion products of the 5′ exonucleases of DNAPTaq and DNAPEc1, shortoligonucleotides (≦12 nucleotides) can also be observed implying thatthese so-called 5′ exonucleases can function endonucleolytically[Setlow, supra; Holland et al., Proc. Natl. Acad. Sci. USA 88:7276(1991)].

In WO 92/06200, Gelfand et al. show that the preferred substrate of the5′ exonuclease activity of the thermostable DNA polymerases is displacedsingle-stranded DNA. Hydrolysis of the phosphodiester bond occursbetween the displaced single-standed DNA and the double-helical DNA withthe preferred exonuclease cleavage site being a phosphodiester bond inthe double helical region. Thus, the 5′ exonuclease activity usuallyassociated with DNAPs is a structure-dependent single-strandedendonuclease and is more properly referred to as a 5′ nuclease.Exonucleases are enzymes which cleave nucleotide molecules from the endsof the nucleic acid molecule. Endonucleases, on the other hand, areenzymes which cleave the nucleic acid molecule at internal rather thanterminal sites. The nuclease activity associated with some thermostableDNA polymerases cleaves endonucleolytically but this cleavage requirescontact with the 5′ end of the molecule being cleaved. Therefore, thesenucleases are referred to as 5′ nucleases.

When a 5′ nuclease activity is associated with a eubacterial Type A DNApolymerase, it is found in the one-third N-terminal region of theprotein as an independent functional domain. The C-terminal two-thirdsof the molecule constitute the polymerization domain which isresponsible for the synthesis of DNA. Some Type A DNA polymerases alsohave a 3′ exonuclease activity associated with the two-third C-terminalregion of the molecule.

The 5′ exonuclease activity and the polymerization activity of DNAPshave been separated by proteolytic cleavage or genetic manipulation ofthe polymerase molecule. To date thermostable DNAPs have been modifiedto remove or reduce the amount of 5′ nuclease activity while leaving thepolymerase activity intact.

The Klenow or large proteolytic cleavage fragment of DNAPEc1 containsthe polymerase and 3′ exonuclease activity but lacks the 5′ nucleaseactivity. The Stoffel fragment of DNAPTaq (DNAPStf) lacks the 5′nuclease activity due to a genetic manipulation which deleted theN-terminal 289 amino acids of the polymerase molecule [Erlich et al.,Science 252:1643 (1991)]. WO 92/06200 describes a thermostable DNAP withan altered level of 5′ to 3′ exonuclease. U.S. Pat. No. 5,108,892describes a Thermus aquaticus DNAP without a 5′ to 3′ exonuclease.However, the art of molecular biology lacks a thermostable DNApolymerase with a lessened amount of synthetic activity.

The present invention provides 5′ nucleases derived from thermostableType A DNA polymerases that retain 5′ nuclease activity but have reducedor absent synthetic activity. The ability to uncouple the syntheticactivity of the enzyme from the 5′ nuclease activity proves that the 5′nuclease activity does not require concurrent DNA synthesis as waspreviously reported (Gelfand, PCR Technology, supra).

The description of the invention is divided into: I. Detection ofSpecific Nucleic Acid Sequences Using 5′ Nucleases; II. Generation of 5′Nucleases Derived From Thermostable DNA Polymerases; III. Detection ofSpecific Nucleic Acid Sequences Using 5′ Nucleases in anInvader-Directed Cleavage Assay; IV. A Comparison of Invasive CleavageAnd Primer-Directed Cleavage; V. Fractionation of Specific Nucleic AcidsBy Selective Charge Reversal; VI. Invader™-Directed Cleavage UsingMiniprobes And Mid-Range Probes; VII. Signal Enhancement By Tailing ofReaction Products In The Invader™-Directed Cleavage Assay; VIII.Improved Enzymes For Use In Invader™-Directed Cleavage Reactions

I. Detection of Specific Nucleic Acid Sequences Using 5′ Nucleases

The 5′ nucleases of the invention form the basis of a novel detectionassay for the identification of specific nucleic acid sequences. Thisdetection system identifies the presence of specific nucleic acidsequences by requiring the annealing of two oligonucleotide probes totwo portions of the target sequence. As used herein, the term “targetsequence” or “target nucleic acid sequence” refers to a specific nucleicacid sequence within a polynucleotide sequence, such as genomic DNA orRNA, which is to be either detected or cleaved or both.

FIG. 1A provides a schematic of one embodiment of the detection methodof the present invention. The target sequence is recognized by twodistinct oligonucleotides in the triggering or trigger reaction. It ispreferred that one of these oligonucleotides is provided on a solidsupport. The other can be provided free. In FIG. 1A the free oligo isindicated as a “primer” and the other oligo is shown attached to a beaddesignated as type 1. The target nucleic acid aligns the twooligonucleotides for specific cleavage of the 5′ arm (of the oligo onbead 1) by the DNAPs of the present invention (not shown in FIG. 1A).

The site of cleavage (indicated by a large solid arrowhead) iscontrolled by the distance between the 3′ end of the “primer” and thedownstream fork of the oligo on bead 1. The latter is designed with anuncleavable region (indicated by the striping). In this manner neitheroligonucleotide is subject to cleavage when misaligned or whenunattached to target nucleic acid.

Successful cleavage releases a single copy of what is referred to as thealpha signal oligo. This oligo may contain a detectable moiety (e.g.,fluorescein). On the other hand, it may be unlabelled.

In one embodiment of the detection method, two more oligonucleotides areprovided on solid supports. The oligonucleotide shown in FIG. 1A on bead2 has a region that is complementary to the alpha signal oligo(indicated as alpha prime) allowing for hybridization. This structurecan be cleaved by the DNAPs of the present invention to release the betasignal oligo. The beta signal oligo can then hybridize to type 3 beadshaving an oligo with a complementary region (indicated as beta prime).Again, this structure can be cleaved by the DNAPs of the presentinvention to release a new alpha oligo.

At this point, the amplification has been linear. To increase the powerof the method, it is desired that the alpha signal oligo hybridized tobead type 2 be liberated after release of the beta oligo so that it maygo on to hybridize with other oligos on type 2 beads. Similarly, afterrelease of an alpha oligo from type 3 beads, it is desired that the betaoligo be liberated.

The liberation of “captured” signal oligos can be achieved in a numberof ways. First, it has been found that the DNAPs of the presentinvention have a true 5′ exonuclease capable of “nibbling” the 5′ end ofthe alpha (and beta) prime oligo (discussed below in more detail). Thus,under appropriate conditions, the hybridization is destabilized bynibbling of the DNAP. Second, the alpha-alpha prime (as well as thebeta-beta prime) complex can be destabilized by heat (e.g., thermalcycling).

With the liberation of signal oligos by such techniques, each cleavageresults in a doubling of the number of signal oligos. In this manner,detectable signal can quickly be achieved.

FIG. 1B provides a schematic of a second embodiment of the detectionmethod of the present invention. Again, the target sequence isrecognized by two distinct oligonucleotides in the triggering or triggerreaction and the target nucleic acid aligns the two oligonucleotides forspecific cleavage of the 5′ arm by the DNAPs of the present invention(not shown in FIG. 1B). The first oligo is completely complementary to aportion of the target sequence. The second oligonucleotide is partiallycomplementary to the target sequence; the 3′ end of the secondoligonucleotide is fully complementary to the target sequence while the5′ end is non-complementary and forms a single-stranded arm. Thenon-complementary end of the second oligonucleotide may be a genericsequence which can be used with a set of standard hairpin structures(described below). The detection of different target sequences wouldrequire unique portions of two oligonucleotides: the entire firstoligonucleotide and the 3′ end of the second oligonucleotide. The 5′ armof the second oligonucleotide can be invariant or generic in sequence.

The annealing of the first and second oligonucleotides near one anotheralong the target sequence forms a forked cleavage structure which is asubstrate for the 5′ nuclease of DNA polymerases. The approximatelocation of the cleavage site is again indicated by the large solidarrowhead in FIG. 1B.

The 5′ nucleases of the invention are capable of cleaving this structurebut are not capable of polymerizing the extension of the 3′ end of thefirst oligonucleotide. The lack of polymerization activity isadvantageous as extension of the first oligonucleotide results indisplacement of the annealed region of the second oligonucleotide andresults in moving the site of cleavage along the second oligonucleotide.If polymerization is allowed to occur to any significant amount,multiple lengths of cleavage product will be generated. A singlecleavage product of uniform length is desirable as this cleavage productinitiates the detection reaction.

The trigger reaction may be run under conditions that allow forthermocycling. Thermocycling of the reaction allows for a logarithicincrease in the amount of the trigger oligonucleotide released in thereaction.

The second part of the detection method allows the annealing of thefragment of the second oligonucleotide liberated by the cleavage of thefirst cleavage structure formed in the triggering reaction (called thethird or trigger oligonucleotide) to a first hairpin structure. Thisfirst hairpin structure has a single-stranded 5′ arm and asingle-stranded 3′ arm. The third oligonucleotide triggers the cleavageof this first hairpin structure by annealing to the 3′ arm of thehairpin thereby forming a substrate for cleavage by the 5′ nuclease ofthe present invention. The cleavage of this first hairpin structuregenerates two reaction products: 1) the cleaved 5′ arm of the hairpincalled the fourth oligonucleotide, and 2) the cleaved hairpin structurewhich now lacks the 5′ arm and is smaller in size than the uncleavedhairpin. This cleaved first hairpin may be used as a detection moleculeto indicate that cleavage directed by the trigger or thirdoligonucleotide occurred. Thus, this indicates that the first twooligonucleotides found and annealed to the target sequence therebyindicating the presence of the target sequence in the sample.

The detection products are amplified by having the fourtholigonucleotide anneal to a second hairpin structure. This hairpinstructure has a 5′ single-stranded arm and a 3′ single-stranded arm. Thefourth oligonucleotide generated by cleavage of the first hairpinstructure anneals to the 3′ arm of the second hairpin structure therebycreating a third cleavage structure recognized by the 5′ nuclease. Thecleavage of this second hairpin structure also generates two reactionproducts: 1) the cleaved 5′ arm of the hairpin called the fiftholigonucleotide which is similar or identical in sequence to the thirdnucleotide, and 2) the cleaved second hairpin structure which now lacksthe 5′ arm and is smaller in size than the uncleaved hairpin. Thiscleaved second hairpin may be as a detection molecule and amplifies thesignal generated by the cleavage of the first hairpin structure.Simultaneously with the annealing of the forth oligonucleotide, thethird oligonucleotide is dissociated from the cleaved first hairpinmolecule so that it is free to anneal to a new copy of the first hairpinstructure. The disassociation of the oligonucleotides from the hairpinstructures may be accomplished by heating or other means suitable todisrupt base-pairing interactions.

Further amplification of the detection signal is achieved by annealingthe fifth oligonucleotide (similar or identical in sequence to the thirdoligonucleotide) to another molecule of the first hairpin structure.Cleavage is then performed and the oligonucleotide that is liberatedthen is annealed to another molecule of the second hairpin structure.Successive rounds of annealing and cleavage of the first and secondhairpin structures, provided in excess, are performed to generate asufficient amount of cleaved hairpin products to be detected. Thetemperature of the detection reaction is cycled just below and justabove the annealing temperature for the oligonucleotides used to directcleavage of the hairpin structures, generally about 55° C. to 70° C. Thenumber of cleavages will double in each cycle until the amount ofhairpin structures remaining is below the K_(m) for the hairpinstructures. This point is reached when the hairpin structures aresubstantially used up. When the detection reaction is to be used in aquantitative manner, the cycling reactions are stopped before theaccumulation of the cleaved hairpin detection products reach a plateau.

Detection of the cleaved hairpin structures may be achieved in severalways. In one embodiment detection is achieved by separation on agaroseor polyacrylamide gels followed by staining with ethidium bromide. Inanother embodiment, detection is achieved by separation of the cleavedand uncleaved hairpin structures on a gel followed by autoradiographywhen the hairpin structures are first labelled with a radioactive probeand separation on chromatography columns using HPLC or FPLC followed bydetection of the differently sized fragments by absorption at OD₂₆₀.Other means of detection include detection of changes in fluorescencepolarization when the single-stranded 5′ arm is released by cleavage,the increase in fluorescence of an intercalating fluorescent indicatoras the amount of primers annealed to 3′ arms of the hairpin structuresincreases. The formation of increasing amounts of duplex DNA (betweenthe primer and the 3′ arm of the hairpin) occurs if successive rounds ofcleavage occur.

The hairpin structures may be attached to a solid support, such as anagarose, styrene or magnetic bead, via the 3′ end of the hairpin. Aspacer molecule may be placed between the 3′ end of the hairpin and thebead, if so desired. The advantage of attaching the hairpin structuresto a solid support is that this prevents the hybridization of the twohairpin structures to one another over regions which are complementary.If the hairpin structures anneal to one another, this would reduce theamount of hairpins available for hybridization to the primers releasedduring the cleavage reactions. If the hairpin structures are attached toa solid support, then additional methods of detection of the products ofthe cleavage reaction may be employed. These methods include, but arenot limited to, the measurement of the released single-stranded 5′ armwhen the 5′ arm contains a label at the 5′ terminus. This label may beradioactive, fluorescent, biotinylated, etc. If the hairpin structure isnot cleaved, the 5′ label will remain attached to the solid support. Ifcleavage occurs, the 5′ label will be released from the solid support.

The 3′ end of the hairpin molecule may be blocked through the use ofdideoxynucleotides. A 3′ terminus containing a dideoxynucleotide isunavailable to participate in reactions with certain DNA modifyingenzymes, such as terminal transferase. Cleavage of the hairpin having a3′ terminal dideoxynucleotide generates a new, unblocked 3′ terminus atthe site of cleavage. This new 3′ end has a free hydroxyl group whichcan interact with terminal transferase thus providing another means ofdetecting the cleavage products.

The hairpin structures are designed so that their self-complementaryregions are very short (generally in the range of 3-8 base pairs). Thus,the hairpin structures are not stable at the high temperatures at whichthis reaction is performed (generally in the range of 50-75° C.) unlessthe hairpin is stabilized by the presence of the annealedoligonucleotide on the 3′ arm of the hairpin. This instability preventsthe polymerase from cleaving the hairpin structure in the absence of anassociated primer thereby preventing false positive results due tonon-oligonucleotide directed cleavage.

As discussed above, the use of the 5′ nucleases of the invention whichhave reduced polymerization activity is advantageous in this method ofdetecting specific nucleic acid sequences. Significant amounts ofpolymerization during the cleavage reaction would cause shifting of thesite of cleavage in unpredictable ways resulting in the production of aseries of cleaved hairpin structures of various sizes rather than asingle easily quantifiable product. Additionally, the primers used inone round of cleavage could, if elongated, become unusable for the nextcycle, by either forming an incorrect structure or by being too long tomelt off under moderate temperature cycling conditions. In a pristinesystem (i.e., lacking the presence of dNTPs), one could use theunmodified polymerase, but the presence of nucleotides (dNTPs) candecrease the per cycle efficiency enough to give a false negativeresult. When a crude extract (genomic DNA preparations, crude celllysates, etc.) is employed or where a sample of DNA from a PCR reaction,or any other sample that might be contaminated with dNTPs, the 5′nucleases of the present invention that were derived from thermostablepolymerases are particularly useful.

II. Generation of 5′ Nucleases From Thermostable DNA Polymerases

The genes encoding Type A DNA polymerases share about 85% homology toeach other on the DNA sequence level. Preferred examples of thermostablepolymerases include those isolated from Thermus aquaticus, Thermusflavus, and Thermus thermophilus. However, other thermostable Type Apolymerases which have 5′ nuclease activity are also suitable. FIGS. 2and 3 compare the nucleotide and amino acid sequences of the three abovementioned polymerases. In FIGS. 2 and 3, the consensus or majoritysequence derived from a comparison of the nucleotide (FIG. 2) or aminoacid (FIG. 3) sequence of the three thermostable DNA polymerases isshown on the top line. A dot appears in the sequences of each of thesethree polymerases whenever an amino acid residue in a given sequence isidentical to that contained in the consensus amino acid sequence. Dashesare used to introduce gaps in order to maximize alignment between thedisplayed sequences. When no consensus nucleotide or amino acid ispresent at a given position, an “X” is placed in the consensus sequence.SEQ ID NOS:1-3 display the nucleotide sequences and SEQ ID NOS:4-6display the amino acid sequences of the three wild-type polymerases. SEQID NO:1 corresponds to the nucleic acid sequence of the wild typeThermus aquaticus DNA polymerase gene isolated from the YT-1 strain[Lawyer et al., J. Biol. Chem. 264:6427 (1989)]. SEQ ID NO:2 correspondsto the nucleic acid sequence of the wild type Thermus flavus DNApolymerase gene [Akhmetzjanov and Vakhitov, Nucl. Acids Res. 20:5839(1992)]. SEQ ID NO:3 corresponds to the nucleic acid sequence of thewild type Thermus thermophilus DNA polymerase gene [Gelfand et al., WO91/09950 (1991)]. SEQ ID NOS:7-8 depict the consensus nucleotide andamino acid sequences, respectively for the above three DNAPs (also shownon the top row in FIGS. 2 and 3).

The 5′ nucleases of the invention derived from thermostable polymeraseshave reduced synthetic ability, but retain substantially the same 5′exonuclease activity as the native DNA polymerase. The term“substantially the same 5′ nuclease activity” as used herein means thatthe 5′ nuclease activity of the modified enzyme retains the ability tofunction as a structure-dependent single-stranded endonuclease but notnecessarily at the same rate of cleavage as compared to the unmodifiedenzyme. Type A DNA polymerases may also be modified so as to produce anenzyme which has increases 5′ nuclease activity while having a reducedlevel of synthetic activity. Modified enzymes having reduced syntheticactivity and increased 5′ nuclease activity are also envisioned by thepresent invention.

By the term “reduced synthetic activity” as used herein it is meant thatthe modified enzyme has less than the level of synthetic activity foundin the unmodified or “native” enzyme. The modified enzyme may have nosynthetic activity remaining or may have that level of syntheticactivity that will not interfere with the use of the modified enzyme inthe detection assay described below. The 5′ nucleases of the presentinvention are advantageous in situations where the cleavage activity ofthe polymerase is desired, but the synthetic ability is not (such as inthe detection assay of the invention).

As noted above, it is not intended that the invention be limited by thenature of the alteration necessary to render the polymerase synthesisdeficient. The present invention contemplates a variety of methods,including but not limited to:

1) proteolysis; 2) recombinant constructs (including mutants); and 3)physical and/or chemical modification and/or inhibition.

1. Proteolysis

Thermostable DNA polymerases having a reduced level of syntheticactivity are produced by physically cleaving the unmodified enzyme withproteolytic enzymes to produce fragments of the enzyme that aredeficient in synthetic activity but retain 5′ nuclease activity.Following proteolytic digestion, the resulting fragments are separatedby standard chromatographic techniques and assayed for the ability tosynthesize DNA and to act as a 5′ nuclease. The assays to determinesynthetic activity and 5′ nuclease activity are described below.

2. Recombinant Constructs

The examples below describe a preferred method for creating a constructencoding a 5′ nuclease derived from a thermostable DNA polymerase. Asthe Type A DNA polymerases are similar in DNA sequence, the cloningstrategies employed for the Thermus aquaticus and flavus polymerases areapplicable to other thermostable Type A polymerases. In general, athermostable DNA polymerase is cloned by isolating genomic DNA usingmolecular biological methods from a bacteria containing a thermostableType A DNA polymerase. This genomic DNA is exposed to primers which arecapable of amplifying the polymerase gene by PCR

This amplified polymerase sequence is then subjected to standarddeletion processes to delete the polymerase portion of the gene.Suitable deletion processes are described below in the examples.

The example below discusses the strategy used to determine whichportions of 54the DNAPTaq polymerase domain could be removed withouteliminating the 5′ nuclease activity. Deletion of amino acids from theprotein can be done either by deletion of the encoding genetic material,or by introduction of a translational stop codon by mutation or frameshift. In addition, proteolytic treatment of the protein molecule can beperformed to remove segments of the protein.

In the examples below, specific alterations of the Taq gene were: adeletion between nucleotides 1601 and 2502 (the end of the codingregion), a 4 nucleotide insertion at position 2043, and deletionsbetween nucleotides 1614 and 1848 and between nucleotides 875 and 1778(numbering is as in SEQ ID NO:1). These modified sequences are describedbelow in the examples and at SEQ ID NOS:9-12.

Those skilled in the art understand that single base pair changes can beinnocuous in terms of enzyme structure and function. Similarly, smalladditions and deletions can be present without substantially changingthe exonuclease or polymerase function of these enzymes.

Other deletions are also suitable to create the 5′ nucleases of thepresent invention. It is preferable that the deletion decrease thepolymerase activity of the 5′ nucleases to a level at which syntheticactivity will not interfere with the use of the 5′ nuclease in thedetection assay of the invention. Most preferably, the synthetic abilityis absent. Modified polymerases are tested for the presence of syntheticand 5′ nuclease activity as in assays described below. Thoughtfulconsideration of these assays allows for the screening of candidateenzymes whose structure is heretofore as yet unknown. In other words,construct “X” can be evaluated according to the protocol described belowto determine whether it is a member of the genus of 5′ nucleases of thepresent invention as defined functionally, rather than structurally.

In the example below, the PCR product of the amplified Thermus aquaticusgenomic DNA did not have the identical nucleotide structure of thenative genomic DNA and did not have the same synthetic ability of theoriginal clone. Base pair changes which result due to the infidelity ofDNAPTaq during PCR amplification of a polymerase gene are also a methodby which the synthetic ability of a polymerase gene may be inactivated.The examples below and FIGS. 4A and 5A indicate regions in the nativeThermus aquaticus and flavus DNA polymerases likely to be important forsynthetic ability. There are other base pair changes and substitutionsthat will likely also inactivate the polymerase.

It is not necessary, however, that one start out the process ofproducing a 5′ nuclease from a DNA polymerase with such a mutatedamplified product. This is the method by which the examples below wereperformed to generate the synthesis-deficient DNAPTaq mutants, but it isunderstood by those skilled in the art that a wild-type DNA polymerasesequence may be used as the starting material for the introduction ofdeletions, insertion and substitutions to produce a 5′ nuclease. Forexample, to generate the synthesis-deficient DNAPTfl mutant, the primerslisted in SEQ ID NOS:13-14 were used to amplify the wild type DNApolymerase gene from Thermus flavus strain AT-62. The amplifiedpolymerase gene was then subjected to restriction enzyme digestion todelete a large portion of the domain encoding the synthetic activity.

The present invention contemplates that the nucleic acid construct ofthe present invention be capable of expression in a suitable host. Thosein the art know methods for attaching various promoters and 3′ sequencesto a gene structure to achieve efficient expression. The examples belowdisclose two suitable vectors and six suitable vector constructs. Ofcourse, there are other promoter/vector combinations that would besuitable. It is not necessary that a host organism be used for theexpression of the nucleic acid constructs of the invention. For example,expression of the protein encoded by a nucleic acid construct may beachieved through the use of a cell-free in vitrotranscription/translation system. An example of such a cell-free systemis the commercially available TnT™ Coupled Reticulocyte Lysate System(Promega Corporation, Madison, Wis.).

Once a suitable nucleic acid construct has been made, the 5′ nucleasemay be produced from the construct. The examples below and standardmolecular biological teachings enable one to manipulate the construct bydifferent suitable methods.

Once the 5′ nuclease has been expressed, the polymerase is tested forboth synthetic and nuclease activity as described below.

3. Physical And/Or Chemical Modification And/Or Inhibition

The synthetic activity of a thermostable DNA polymerase may be reducedby chemical and/or physical means. In one embodiment, the cleavagereaction catalyzed by the 5′ nuclease activity of the polymerase is rununder conditions which preferentially inhibit the synthetic activity ofthe polymerase. The level of synthetic activity need only be reduced tothat level of activity which does not interfere with cleavage reactionsrequiring no significant synthetic activity.

As shown in the examples below, concentrations of Mg⁺⁺ greater than 5 mMinhibit the polymerization activity of the native DNAPTaq. The abilityof the 5′ nuclease to function under conditions where synthetic activityis inhibited is tested by running the assays for synthetic and 5′nuclease activity, described below, in the presence of a range of Mg⁺⁺concentrations (5 to 10 mM). The effect of a given concentration of Mg⁺⁺is determined by quantitation of the amount of synthesis and cleavage inthe test reaction as compared to the standard reaction for each assay.

The inhibitory effect of other ions, polyamines, denaturants, such asurea, formamide, dimethylsulfoxide, glycerol and non-ionic detergents(Triton X-100 and Tween-20), nucleic acid binding chemicals such as,actinomycin D, ethidiun bromide and psoralens, are tested by theiraddition to the standard reaction buffers for the synthesis and 5′nuclease assays. Those compounds having a preferential inhibitory effecton the synthetic activity of a thermostable polymerase are then used tocreate reaction conditions under which 5′ nuclease activity (cleavage)is retained while synthetic activity is reduced or eliminated.

Physical means may be used to preferentially inhibit the syntheticactivity of a polymerase. For example, the synthetic activity ofthermostable polymerases is destroyed by exposure of the polymerase toextreme heat (typically 96 to 100° C.) for extended periods of time(greater than or equal to 20 minutes). While these are minor differenceswith respect to the specific heat tolerance for each of the enzymes,these are readily determined. Polymerases are treated with heat forvarious periods of time and the effect of the heat treatment upon thesynthetic and 5′ nuclease activities is determined.

III. Detection of Specific Nucleic Acid Sequences Using 5′ Nucleases InAn Invader-Directed Cleavage Assay

The present invention provides means for forming a nucleic acid cleavagestructure which is dependent upon the presence of a target nucleic acidand cleaving the nucleic acid cleavage structure so as to releasedistinctive cleavage products. 5′ nuclease activity is used to cleavethe target-dependent cleavage structure and the resulting cleavageproducts are indicative of the presence of specific target nucleic acidsequences in the sample.

The present invention further provides assays in which the targetnucleic acid is reused or recycled during multiple rounds ofhybridization with oligonucleotide probes and cleavage without the needto use temperature cycling (i.e., for periodic denaturation of targetnucleic acid strands) or nucleic acid synthesis (i.e., for thedisplacement of target nucleic acid strands). Through the interaction ofthe cleavage means (e.g., a 5′ nuclease) an upstream oligonucleotide,the cleavage means can be made to cleave a downstream oligonucleotide atan internal site in such a way that the resulting fragments of thedownstream oligonucleotide dissociate from the target nucleic acid,thereby maling that region of the target nucleic acid available forhybridization to another, uncleaved copy of the downstreamoligonucleotide.

As illustrated in FIG. 29, the methods of the present invention employat least a pair of oligonucleotides that interact with a target nucleicacid to form a cleavage structure for a structure-specific nuclease.More specifically, the cleavage structure comprises i) a target nucleicacid that may be either single-stranded or double-stranded (when adouble-stranded target nucleic acid is employed, it may be renderedsingle stranded, e.g., by heating); ii) a first oligonucleotide, termedthe “probe,” which defines a first region of the target nucleic acidsequence by being the complement of that region (regions X and Z of thetarget as shown in FIG. 29); iii) a second oligonucleotide, termed the“invader,” the 5′ part of which defines a second region of the sametarget nucleic acid sequence (regions Y and X in FIG. 29), adjacent toand downstream of the first target region (regions X and Z), and thesecond part of which overlaps into the region defined by the firstoligonucleotide (region X depicts the region of overlap). The resultingstructure is diagrammed in FIG. 29.

While not limiting the invention or the instant discussion to anyparticular mechanism of action, the diagram in FIG. 29 represents theeffect on the site of cleavage caused by this type of arrangement of apair of oligonucleotides. The design of such a pair of oligonucleotidesis described below in detail. In FIG. 29, the 3′ ends of the nucleicacids (i.e., the target and the oligonucleotides) are indicated by theuse of the arrowheads on the ends of the lines depicting the strands ofthe nucleic acids (and where space permits, these ends are also labelled“3′”). It is readily appreciated that the two oligonucleotides (theinvader and the probe) are arranged in a parallel orientation relativeto one another, while the target nucleic acid strand is arranged in ananti-parallel orientation relative to the two oligonucleotides. Furtherit is clear that the invader oligonucleotide is located upstream of theprobe oligonucleotide and that with respect to the target nucleic acidstrand, region Z is upstream of region X and region X is upstream ofregion Y (that is region Y is downstream of region X and region X isdownstream of region Z). Regions of complementarity between the opposingstrands are indicated by the short vertical lines. While not intended toindicate the precise location of the site(s) of cleavage, the area towhich the site of cleavage within the probe oligonucleotide is shiftedby the presence of the invader oligonucleotide is indicated by the solidvertical arrowhead. An alternative representation of thetarget/invader/probe cleavage structure is shown in FIG. 32 c. Neitherdiagram (i.e., FIG. 29 or FIG. 32 c) is intended to represent the actualmechanism of action or physical arrangement of the cleavage structureand further it is not intended that the method of the present inventionbe limited to any particular mechanism of action.

It can be considered that the binding of these oligonucleotides dividesthe target nucleic acid into three distinct regions: one region that hascomplementarity to only the probe (shown as “Z”); one region that hascomplementarity only to the invader (shown as “Y”); and one region thathas complementarity to both oligonucleotides (shown as “X”).

Design of these oligonucleotides (ie., the invader and the probe) isaccomplished using practices which are standard in the art. For example,sequences that have self complementarity, such that the resultingoligonucleotides would either fold upon themselves, or hybridize to eachother at the expense of binding to the target nucleic acid, aregenerally avoided.

One consideration in choosing a length for these oligonucleotides is thecomplexity of the sample containing the target nucleic acid. Forexample, the human genome is approximately 3×10⁹ basepairs in length.Any 10 nucleotide sequence will appear with a frequency of 1:4¹⁰, or1:1048,576 in a random string of nucleotides, which would beapproximately 2,861 times in 3 billion basepairs. Clearly anoligonucleotide of this length would have a poor chance of bindinguniquely to a 10 nucleotide region within a target having a sequence thesize of the human genome. If the target sequence were within a 3 kbplasmid, however, such an oligonucleotide might have a very reasonablechance of binding uniquely. By this same calculation it can be seen thatan oligonucleotide of 16 nucleotides (i.e., a 16-mer) is the minimumlength of a sequence which is mathematically likely to appear once in3×10⁹ basepairs.

A second consideration in choosing oligonucleotide length is thetemperature range in which the oligonucleotides will be expected tofunction. A 16-mer of average base content (50% G-C basepairs) will havea calculated T_(m) (the temperature at which 50% of the sequence isdissociated) of about 41° C., depending on, among other things, theconcentration of the oligonucleotide and its target, the salt content ofthe reaction and the precise order of the nucleotides. As a practicalmatter, longer oligonucleotides are usually chosen to enhance thespecificity of hybridization. Oligonucleotides 20 to 25 nucleotides inlength are often used as they are highly likely to be specific if usedin reactions conducted at temperatures which are near their T_(m)s(within about 5° of the T_(m)). In addition, with calculated T_(m)s inthe range of 50° to 70° C., such oligonucleotides (i.e, 20 to 25-mers)are appropriately used in reactions catalyzed by thermostable enzymes,which often display optimal activity near this temperature range.

The maximum length of the oligonucleotide chosen is also based on thedesired specificity. One must avoid choosing sequences that are so longthat they are either at a high risk of binding stably to partialcomplements, or that they cannot easily be dislodged when desired (e.g.,failure to disassociate from the target once cleavage has occurred).

The first step of design and selection of the oligonucleotides for theinvader-directed cleavage is in accordance with these sample generalprinciples. Considered as sequence-specific probes individually, eacholigonucleotide may be selected according to the guidelines listedabove. That is to say, each oligonucleotide will generally be longenough to be reasonably expected to hybridize only to the intendedtarget sequence within a complex sample, usually in the 20 to 40nucleotide range. Alternatively, because the invader-directed cleavageassay depends upon the concerted action of these oligonucleotides, thecomposite length of the 2 oligonucleotides which span/bind to the X, Y,Z regions may be selected to fall within this range, with each of theindividual oligonucleotides being in approximately the 13 to 17nucleotide range. Such a design might be employed if a non-thermostablecleavage means were employed in the reaction, requiring the reactions tobe conducted at a lower temperature than that used when thermostablecleavage means are employed. In some instances, it may be desirable tohave these oligonucleotides bind multiple times within a target nucleicacid (e.g., which bind to multiple variants or multiple similarsequences within a target). It is not intended that the method of thepresent invention be limited to any particular size of the probe orinvader oligonucleotide.

The second step of designing an oligonucleotide pair for this assay isto choose the degree to which the upstream “invader” oligonucleotidesequence will overlap into the downstream “probe” oligonucleotidesequence, and consequently, the sizes into which the probe will becleaved. A key feature of this assay is that the probe oligonucleotidecan be made to “turn over,” that is to say cleaved probe can be made todepart to allow the binding and cleavage of other copies of the probemolecule, without the requirements of thermal denaturation ordisplacement by polymerization.

While in one embodiment of this assay probe turnover may be facilitatedby an exonucleolytic digestion by the cleavage agent, it is central tothe present invention that the turnover does not require thisexonucleolytic activity.

Choosing The Amount of Overlap (Length of The X Region)

One way of accomplishing such turnover can be envisioned by consideringthe diagram in FIG. 29. It can be seen that the Tm of eacholigonucleotide will be a function of the full length of thatoligonucleotide: i.e., the Tm of the invader=Tm(Y+X), and the Tm of theprobe=Tm_((X+Y)) for the probe. When the probe is cleaved the X regionis released, leaving the Z section. If the Tm of Z is less than thereaction temperature, and the reaction temperature is less than theTm_((X+Z)), then cleavage of the probe will lead to the departure of Z,thus allowing a new (X+Z) to hybridize. It can be seen from this examplethat the X region must be sufficiently long that the release of X willdrop the Tm of the remaining probe section below the reactiontemperature: a G-C rich X section may be much shorter than an A-T rich Xsection and still accomplish this stability shift.

Designing Oligonucleotides Which Interact With The Y And Z Regions

If the binding of the invader oligonucleotide to the target is morestable than the binding of the probe (e.g., if it is long, or is rich inG-C basepairs in the Y region), then the copy of X associated with theinvader may be favored in the competition for binding to the X region ofthe target, and the probe may consequently hybridize inefficiently, andthe assay may give low signal. Alternatively, if the probe binding isparticularly strong in the Z region, the invader will still causeinternal cleavage, because this is mediated by the enzyme, but portionof the probe oligonucleotide bound to the Z region may not dissociate atthe reaction temperature, turnover may be poor, and the assay may againgive low signal.

It is clearly beneficial for the portions of the oligonucleotide whichinteract with the Y and Z regions so be similar in stability, i.e., theymust have similar melting temperatures. This is not to say that theseregions must be the same length. As noted above, in addition to length,the melting temperature will also be affected by the base content andthe specific sequence of those bases. The specific stability designedinto the invader and probe sequences will depend on the temperature atwhich one desires to perform the reaction.

This discussion is intended to illustrate that (within the basicguidelines for oligonucleotide specificity discussed above) it is thebalance achieved between the stabilities of the probe and invadersequences and their X and Y component sequences, rather than theabsolute values of these stabilities, that is the chief consideration inthe selection of the probe and invader sequences.

Design of The Reaction Conditions

Target nucleic acids that may be analyzed using the methods of thepresent invention which employ a 5′ nuclease as the cleavage meansinclude many types of both RNA and DNA. Such nucleic acids may beobtained using standard molecular biological techniques. For example,nucleic acids (RNA or DNA) may be isolated from a tissue sample (e.g, abiopsy specimen), tissue culture cells, samples containing bacteriaand/or viruses (including cultures of bacteria and/or viruses), etc. Thetarget nucleic acid may also be transcribed in vitro from a DNA templateor may be chemically synthesized or generated in a PCR. Furthermore,nucleic acids may be isolated from an organism, either as genomicmaterial or as a plasmid or similar extrachromosomal DNA, or they may bea fragment of such material generated by treatment with a restrictionendonuclease or other cleavage agents or it may be synthetic.

Assembly of the target, probe, and invader nucleic acids into thecleavage reaction of the present invention uses principles commonly usedin the design of oligonucleotide base enzymatic assays, such asdideoxynucleotide sequencing and polymerase chain reaction (PCR). As isdone in these assays, the oligonucleotides are provided in sufficientexcess that the rate of hybridization to the target nucleic acid is veryrapid. These assays are commonly performed with 50 fmoles to 2 pmoles ofeach oligonucleotide per μl of reaction mixture. In the Examplesdescribed herein, amounts of oligonucleotides ranging from 250 fmoles to5 pmoles per μl of reaction volume were used. These values were chosenfor the purpose of ease in demonstration and are not intended to limitthe performance of the present invention to these concentrations. Other(e.g., lower) oligonucleotide concentrations commonly used in othermolecular biological reactions are also contemplated.

It is desirable that an invader oligonucleotide be immediately availableto direct the cleavage of each probe oligonucleotide that hybridizes toa target nucleic acid. For this reason, in the Examples describedherein, the invader oligonucleotide is provided in excess over the probeoligonucleotide; often this excess is 10-fold. While this is aneffective ratio, it is not intended that the practice of the presentinvention be limited to any particular ratio of invader-to-probe (aratio of 2- to 100-fold is contemplated).

Buffer conditions must be chosen that will be compatible with both theoligonucleotide/target hybridization and with the activity of thecleavage agent. The optimal buffer conditions for nucleic acidmodification enzymes, and particularly DNA modification enzymes,generally included enough mono- and di-valent salts to allow associationof nucleic acid strands by base-pairing. If the method of the presentinvention is performed using an enzymatic cleavage agent other thanthose specifically described here, the reactions may generally beperformed in any such buffer reported to be optimal for the nucleasefunction of the cleavage agent. In general, to test the utility of anycleavage agent in this method, test reactions are performed wherein thecleavage agent of interest is tested in the MOPS/MnCl₂/KCl buffer orMg-containing buffers described herein and in whatever buffer has beenreported to be suitable for use with that agent, in a manufacturer'sdata sheet, a journal article, or in personal communication.

The products of the invader-directed cleavage reaction are fragmentsgenerated by structure-specific cleavage of the input oligonucleotides.The resulting cleaved and/or uncleaved oligonucleotides may be analyzedand resolved by a number of methods including electrophoresis (on avariety of supports including acrylamide or agarose gels, paper, etc.),chromatography, fluorescence polarization, mass spectrometry and chiphybridization. The invention is illustrated using electrophoreticseparation for the analysis of the products of the cleavage reactions.However, it is noted that the resolution of the cleavage products is notlimited to electrophoresis. Electrophoresis is chosen to illustrate themethod of the invention because electrophoresis is widely practiced inthe art and is easily accessible to the average practioner.

The probe and invader oligonucleotides may contain a label to aid intheir detection following the cleavage reaction. The label may be aradioisotope (e.g., a ³²P or ³⁵S-labelled nucleotide) placed at eitherthe 5′ or 3′ end of the oligonucleotide or alternatively, the label maybe distributed throughout the oligonucleotide (i.e., a uniformlylabelled oligonucleotide). The label may be a nonisotopic detectablemoiety, such as a fluorophore, which can be detected directly,or areactive group which permits specific recognition by a secondary agent.For example, biotinylated oligonucleotides may be detected by probingwith a streptavidin molecule which is coupled to an indicator (e.g.,alkaline phosphatase or a fluorophore) or a hapten such as dioxigeninmay be detected using a specific antibody coupled to a similarindicator.

Optimization of Reaction Conditions

The invader-directed cleavage reaction is useful to detect the presenceof specific nucleic acids. In addition to the considerations listedabove for the selection and design of the invader and probeoligonucleotides, the conditions under which the reaction is to beperformed may be optimized for detection of a specific target sequence.

One objective in optimizing the invader-directed cleavage assay is toallow specific detection of the fewest copies of a target nucleic acid.To achieve this end, it is desirable that the combined elements of thereaction interact with the maximum efficiency, so that the rate of thereaction (e.g., the number of cleavage events per minute) is maximized.Elements contributing to the overall efficiency of the reaction includethe rate of hybridization, the rate of cleavage, and the efficiency ofthe release of the cleaved probe.

The rate of cleavage will be a function of the cleavage means chosen,and may be made optimal according to the manufacturer's instructionswhen using commercial preparations of enzymes or as described in theexamples herein. The other elements (rate of hybridization, efficiencyof release) depend upon the execution of the reaction, and optimizationof these elements is discussed below.

Three elements of the cleavage reaction that significantly affect therate of nucleic acid hybridization are the concentration of the nucleicacids, the temperature at which the cleavage reaction is performed andthe concentration of salts and/or other charge-shielding ions in thereaction solution.

The concentrations at which oligonucleotide probes are used in assays ofthis type are well known in the art, and are discussed above. Oneexample of a common approach to optimizing an oligonucleotideconcentration is to choose a starting amount of oligonucleotide forpilot tests; 0.01 to 2 μM is a concentration range used in manyoligonucleotide-based assays. When initial cleavage reactions areperformed, the following questions may be asked of the data: Is thereaction performed in the absence of the target nucleic acidsubstantially free of the cleavage product?; Is the site of cleavagespecifically shifted in accordance with the design of the invaderoligonucleotide?; Is the specific cleavage product easily detected inthe presence of the uncleaved probe (or is the amount of uncut materialoverwhelming the chosen visualization method)?

A negative answer to any of these questions would suggest that the probeconcentration is too high, and that a set of reactions using serialdilutions of the probe should be performed until the appropriate amountis identified. Once identified for a given target nucleic acid in a givesample type (e.g., purified genomic DNA, body fluid extract, lysedbacterial extract), it should not need to be re-optimized. The sampletype is important because the complexity of the material present mayinfluence the probe optimum.

Conversely, if the chosen initial probe concentration is too low, thereaction may be slow, due to inefficient hybridization. Tests withincreasing quantities of the probe will identify the point at which theconcentration exceeds the optimum. Since the hybridization will befacilitated by excess of probe, it is desirable, but not required, thatthe reaction be performed using probe concentrations just below thispoint.

The concentration of invader oligonucleotide can be chosen based on thedesign considerations discussed above. In a preferred embodiment, theinvader oligonucleotide is in excess of the probe oligonucleotide. In aparticularly preferred embodiment, the invader is approximately 10-foldmore abundant than the probe.

Temperature is also an important factor in the hybridization ofoligonucleotides. The range of temperature tested will depend in largepart, on the design of the oligonucleotides, as discussed above. In apreferred embodiment, the reactions are performed at temperaturesslightly below the T_(m) of the least stable oligonucleotide in thereaction. Melting temperatures for the oligonucleotides and for theircomponent regions (X, Y and Z, FIG. 29), can be estimated through theuse of computer software or, for a more rough approximation, byassigning the value of 2° C. per A-T basepair, and 4° C. per G-Cbasepair, and taking the sum across an expanse of nucleic acid. Thelatter method may be used for oligonucleotides of approximately 10-30nucleotides in length. Because even computer prediction of the T_(m) ofa nucleic acid is only an approximation, the reaction temperatureschosen for initial tests should bracket the calculated T_(m). Whileoptimizations are not limited to this, 5° C. increments are convenienttest intervals in these optimization assays.

When temperatures are tested, the results can be analyzed forspecificity (the first two of the questions listed above) in the sameway as for the oligonucleotide concentration determinations.Non-specific cleavage (i.e., cleavage of the probe at many or allpositions along its length) would indicate non-specific interactionsbetween the probe and the sample material, and would suggest that ahigher temperature should be employed. Conversely, little or no cleavagewould suggest that even the intended hybridization is being prevented,and would suggest the use of lower temperatures. By testing severaltemperatures, it is possible to identify an approximate temperatureoptimum, at which the rate of specific cleavage of the probe is highest.If the oligonucleotides have been designed as described above, the T_(m)of the Z-region of the probe oligonucleotide should be below thistemperature, so that turnover is assured.

A third determinant of hybridization efficiency is the saltconcentration of the reaction. In large part, the choice of solutionconditions will depend on the requirements of the cleavage agent, andfor reagents obtained commercially, the manufacturer's instructions area resource for this information. When developing an assay utilizing anyparticular cleavage agent, the oligonucleotide and temperatureoptimizations described above should be performed in the bufferconditions best suited to that cleavage agent.

A “no enzyme” control allows the assessment of the stability of thelabeled oligonucleotides under particular reaction conditions, or in thepresence of the sample to be tested (i.e., in assessing the sample forcontaminating nucleases). In this manner, the substrate andoligonucleotides are placed in a tube containing all reactioncomponents, except the enzyme and treated the same as theenzyme-containing reactions. Other controls may also be included. Forexample, a reaction with all of the components except the target nucleicacid will serve to confirm the dependence of the cleavage on thepresence of the target sequence.

Probing For Multiple Alleles

The invader-directed cleavage reaction is also useful in the detectionand quantification of individual variants or alleles in a mixed samplepopulation. By way of example, such a need exists in the analysis oftumor material for mutations in genes associated with cancers. Biopsymaterial from a tumor can have a significant complement of normal cells,so it is desirable to detect mutations even when present in fewer than5% of the copies of the target nucleic acid in a sample. In this case,it is also desirable to measure what fraction of the population carriesthe mutation. Similar analyses may also be done to examine allelicvariation in other gene systems, and it is not intended that the methodof the present invention by limited to the analysis of tumors.

As demonstrated below, reactions can be performed under conditions thatprevent the cleavage of probes bearing even a single-nucleotidedifference mismatch within the region of the target nucleic acid termed“Z” in FIG. 29, but that permit cleavage of a similar probe that iscompletely complementary to the target in this region. Thus, the assaymay be used to quantitate individual variants or alleles within a mixedsample.

The use of multiple, differently labelled probes in such an assay isalso contemplated. To assess the representation of different variants oralleles in a sample, one would provide a mixture of probes such thateach allele or variant to be detected would have a specific probe(i.e.,perfectly matched to the Z region of the target sequence) with aunique label (e.g., no two variant probes with the same label would beused in a single reaction). These probes would be characterized inadvance to ensure that under a single set of reaction conditions, theycould be made to give the same rate of signal accumulation when mixedwith their respective target nucleic acids. Assembly of a cleavagereaction comprising the mixed probe set, a corresponding invaderoligonucleotide, the target nucleic acid sample, and the appropriatecleavage agent, along with performance of the cleavage reaction underconditions such that only the matched probes would cleave, would allowindependent quantification of each of the species present, and wouldtherefore indicate their relative representation in the target sample.

IV. A Comparision of Invasive Cleavage And Primer-Directed Cleavage

As discussed herein, the terms “invasive” or “invader-directed” cleavagespecifically denote the use of a first, upstream oligonucleotide, asdefined below, to cause specific cleavage at a site within a second,downstream sequence. To effect such a direction of cleavage to a regionwithin a duplex, it is required that the first and secondoligonucleotides overlap in sequence. That is to say, a portion of theupstream oligonucleotide, termed the “invader”, has significant homologyto a portion of the downstream “probe” oligonucleotide, so that theseregions would tend to basepair with the same complementary region of thetarget nucleic acid to be detected. While not limiting the presentinvention to any particular mechanism, the overlapping regions would beexpected to alternate in their occupation of the shared hybridizationsite. When the probe oligonucleotide fully anneals to the target nucleicacid, and thus forces the 3′ region of the invader to remain unpaired,the structure so formed is not a substrate for the 5′ nucleases of thepresent invention. By contrast, when the inverse is true, the structureso formed is substrate for these enzymes, allowing cleavage and releaseof the portion of the probe oligonucleotide that is displaced by theinvader oligonucleotide. The shifting of the cleavage site to a regionthe probe oligonucleotide that would otherwise be basepaired to thetarget sequence is one hallmark of the invasive cleavage assay (i.e.,the invader-directed cleavage assay) of the present invention.

It is beneficial at this point to contrast the invasive cleavage asdescribed above with two other forms of probe cleavage that may lead tointernal cleavage of a probe oligonucleotide, but which do not compriseinvasive cleavage. In the first case, a hybridized probe may be subjectto duplex-dependent 5′ to 3′ exonuclease “nibbling,” such that theoligonucleotide is shortened from the 5′ end until it cannot remainbound to the target (see, e.g., Examples 6-8 and FIGS. 26-28). The siteat which such nibbling stops can appear to be discrete, and, dependingon the difference between the melting temperature of the fall-lengthprobe and the temperature of the reaction, this stopping point may be 1or several nucleotides into the probe oligonucleotide sequence. Such“nibbling” is often indicated by the presence of a “ladder” of longerproducts ascending size up to that of the full length of the probe, butthis is not always the case. While any one of the products of such anibbling reaction may be made to match in size and cleavage site theproducts of an invasive cleavage reaction, the creation of thesenibbling products would be highly dependent on the temperature of thereaction and the nature of the cleavage agent, but would be independentof the action of an upstream oligonucleotide, and thus could not beconstrued to involve invasive cleavage.

A second cleavage structure that may be considered is one in which aprobe oligonucleotide has several regions of complementarity with thetarget nucleic acid, interspersed with one or more regions ornucleotides of noncomplementarity. These noncomplementary regions may bethought of as “bubbles” within the nucleic acid duplex. As temperatureis elevated, the regions of complementarity can be expected to “melt” inthe order of their stability, lowest to highest. When a region of lowerstability is near the end of a segment of duplex, and the next region ofcomplementarity along the strand has a higher melting temperature, atemperature can be found that will cause the terminal region of duplexto melt first, opening the first bubble, and thereby creating apreferred substrate structure of the cleavage by the 5′ nucleases of thepresent invention (FIG. 40 a). The site of such cleavage would beexpected to be on the 5′ arm, within 2 nucleotides of the junctionbetween the single and double-stranded regions (Lyamichev et al., supra.and U.S. Pat. No. 5,422,253)

An additional oligonucleotide could be introduced to basepair along thetarget nucleic acid would have a similar effect of opening this bubblefor subsequent cleavage of the unpaired 5′ arm (FIG. 40 b and FIG. 6).Note in this case, the 3′ terminal nucleotides of the upstreamoligonucleotide anneals along the target nucleic acid sequence in such amanner that the 3′ end is located within the “bubble” region. Dependingon the precise location of the 3′ end of this oligonucleotide, thecleavage site may be along the newly unpaired 5′ arm, or at the siteexpected for the thermally opened bubble structure as described above.In the former case the cleavage is not within a duplexed region, and isthus not invasive cleavage, while in the latter the oligonucleotide ismerely an aide in inducing cleavage at a site that might otherwise beexposed through the use of temperature alone (i.e., in the absence ofthe additional oligonucleotide), and is thus not considered to beinvasive cleavage.

In summary, any arrangement of oligonucleotides used for thecleavage-based detection of a target sequence can be analyzed todetermine if the arrangement is an invasive cleavage structure ascontemplated herein. An invasive cleavage structure supports cleavage ofthe probe in a region that, in the absence of an upstreamoligonucleotide, would be expected to be basepaired to the targetnucleic acid.

Example 26 below provides further guidance for the design and executionof a experiments which allow the determination of whether a givenarrangement of a pair of upstream and downstream (i.e., the probe)oligonucleotides when annealed along a target nucleic acid would form aninvasive cleavage structure.

V. Fractionation of Specific Nucleic Acids By Selective Charge Reversal

Some nucleic acid-based detection assays involve the elongation and/orshortening of oligonucleotide probes. For example, as described herein,the primer-directed, primer-independent, and invader-directed cleavageassays, as well as the “nibbling” assay all involve the cleavage (i.e.,shortening) of oligonucleotides as a means for detecting the presence ofa target nucleic sequence. Examples of other detection assays whichinvolve the shortening of an oligonucleotide probe include the “TaqMan”or nick-translation PCR assay described in U.S. Pat. No. 5,210,015 toGelfand et al. (the disclosure of which is herein incorporated byreference), the assays described in U.S. Pat. Nos. 4,775,619 and5,118,605 to Urdea (the disclosures of which are herein incorporated byreference), the catalytic hybridization amplification assay described inU.S. Pat. No. 5,403,711 to Walder and Walder (the disclosure of which isherein incorporated by reference), and the cycling probe assay describedin U.S. Pat. Nos. 4,876,187 and 5,011,769 to Duck et al. (thedisclosures of which are herein incorporated by reference). Examples ofdetection assays which involve the elongation of an oligonucleotideprobe (or primer) include the polymerase chain reaction (PCR) describedin U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis and Mullis et al.(the disclosures of which are herein incorporated by reference) and theligase chain reaction (LCR) described in U.S. Pat. Nos. 5,427,930 and5,494,810 to Birkenmeyer et al. and Barany et al. (the disclosures ofwhich are herein incorporated by reference). The above examples areintended to be illustrative of nucleic acid-based detection assays thatinvolve the elongation and/or shortening of oligonucleotide probes anddo not provide an exhaustive list.

Typically, nucleic acid-based detection assays that involve theelongation and/or shortening of oligonucleotide probes requirepost-reaction analysis to detect the products of the reaction. It iscommon that, the specific reaction product(s) must be separated from theother reaction components, including the input or unreactedoligonucleotide probe. One detection technique involves theelectrophoretic separation of the reacted and unreacted oligonucleotideprobe. When the assay involves the cleavage or shortening of the probe,the unreacted product will be longer than the reacted or cleavedproduct. When the assay involves the elongation of the probe (orprimer), the reaction products will be greater in length than the input.Gel-based electrophoresis of a sample containing nucleic acid moleculesof different lengths separates these fragments primarily on the basis ofsize. This is due to the fact that in solutions having a neutral oralkaline pH, nucleic acids having widely different sizes (i.e.,molecular weights) possess very similar charge-to-mass ratios and do notseparate [Andrews, Electrophoresis, 2nd Edition, Oxford University Press(1986), pp. 153-154]. The gel matrix acts as a molecular sieve andallows nucleic acids to be separated on the basis of size and shape(e.g., linear, relaxed circular or covalently closed supercoiledcircles).

Unmodified nucleic acids have a net negative charge due to the presenceof negatively charged phosphate groups contained within thesugar-phosphate backbone of the nucleic acid. Typically, the sample isapplied to gel near the negative pole and the nucleic acid fragmentsmigrate into the gel toward the positive pole with the smallestfragments moving fastest through the gel.

The present invention provides a novel means for fractionating nucleicacid fragments on the basis of charge. This novel separation techniqueis related to the observation that positively charged adducts can affectthe electrophoretic behavior of small oligonucleotides because thecharge of the adduct is significant relative to charge of the wholecomplex. In addition, to the use of positively charged adducts (e.g. Cy3and Cy5 amidite fluorescent dyes, the positively charged heterodimericDNA-binding dyes shown in FIG. 66, etc.), the oligonucleotide maycontain amino acids (particulary useful amino acids are the chargedamino acids: lysine, arginine, asparate, glutamate), modified bases,such as amino-modified bases, and/or a phosphonate backbone (at all or asubset of the positions). In addition as discussed further below, aneutral dye or detection moiety (e.g., biotin, streptavidin, etc.) maybe employed in place of a positively charged adduct in conjunction withthe use of amino-modified bases and/or a complete or partial phosphonatebackbone.

This observed effect is of particular utility in assays based on thecleavage of DNA molecules. Using the assays described herein as anexample, when an oligonucleotide is shortened through the action of aCleavase® enzyme or other cleavage agent, the positive charge can bemade to not only significantly reduce the net negative charge, but toactually override it, effectively “flipping” the net charge of thelabeled entity. This reversal of charge allows the products oftarget-specific cleavage to be partitioned from uncleaved probe byextremely simple means. For example, the products of cleavage can bemade to migrate towards a negative electrode placed at any point in areaction vessel, for focused detection without gel-basedelectrophoresis; Example 24 provides examples of devices suitable forfocused detection without gel-based electrophoresis. When a slab gel isused, sample wells can be positioned in the center of the gel, so thatthe cleaved and uncleaved probes can be observed to migrate in oppositedirections. Alternatively, a traditional vertical gel can be used, butwith the electrodes reversed relative to usual DNA gels (i.e., thepositive electrode at the top and the negative electrode at the bottom)so that the cleaved molecules enter the gel, while the uncleaveddisperse into the upper reservoir of electrophoresis buffer.

An important benefit of this type of readout is the absolute nature ofthe partition of products from substrates, i.e., the separation isvirtually 100%. This means that an abundance of uncleaved probe can besupplied to drive the hybridization step of the probe-based assay, yetthe unconsumed (ie., unreacted) probe can, in essence, be subtractedfrom the result to reduce background by virtue of the fact that theunreacted probe will not migrate to the same pole as the specificreaction product.

Through the use of multiple positively charged adducts, syntheticmolecules can be constructed with sufficient modification that thenormally negatively charged strand is made nearly neutral. When soconstructed, the presence or absence of a single phosphate group canmean the difference between a net negative or a net positive charge.This observation has particular utility when one objective is todiscriminate between enzymatically generated fragments of DNA, whichlack a 3 phosphate, and the products of thermal degradation, whichretain a 3 phosphate (and thus two additional negative charges).Examples 23 and 24 demonstrate the ability to separate positivelycharged reaction products from a net negatively charged substrateoligonucleotide. As discussed in these examples, oligonucleotides may betransformed from net negative to net positively charged compounds. InExample 24, the positively charged dye, Cy3 was incorporated at the 5′end of a 22-mer (SEQ ID NO:61) which also contained twoamino-substituted residues at the 5′ end of the oligonucleotide; thisoligonucleotide probe carries a net negative charge. After cleavage,which occurred 2 nucleotides into the probe, the following labelledoligonucleotide was released: 5′-Cy3-AminoT-AminoT-3′ (as well as theremaining 20 nucleotides of SEQ ID NO:61). This short fragment bears anet positive charge while the reaminder of the cleaved oligonucleotideand the unreacted or input oligonucleotide bear net negative charges.

The present invention contemplates embodiments wherein the specificreaction product produced by any cleavage of any oligonucleotide can bedesigned to carry a net positive charge while the unreacted probe ischarge neutral or carries a net negative charge. The present inventionalso contemplates embodiments where the released product may be designedto carry a net negative charge while the input nucleic acid carries anet positive charge. Depending on the length of the released product tobe detected, positively charged dyes may be incorporated at the one endof the probe and modified bases may be placed along the oligonucleotidesuch that upon cleavage, the released fragment containing the positivelycharged dye carries a net positive charge. Amino-modified bases may beused to balance the charge of the released fragment in cases where thepresence of the positively charged adduct (e.g., dye) alone is notsufficient to impart a net positive charge on the released fragment. Inaddition, the phosphate backbone may be replaced with a phosphonatebackbone at a level sufficient to impart a net positive charge (this isparticularly useful when the sequence of the oligonucleotide is notamenable to the use of amino-substituted bases); FIGS. 56 and 57 showthe structure of short oligonucleotides containing a phosphonate groupon the second T residue). An oligonucleotide containing a fullyphosphonate-substituted backbone would be charge neutral (absent thepresence of modified charged residues bearing a charge or the presenceof a charged adduct) due to the absence of the negatively chargedphosphate groups. Phosphonate-containing nucleotides (e.g.,methylphosphonate-containing nucleotides are readily available and canbe incorporated at any position of an oligonucleotide during synthesisusing techniques which are well known in the art.

In essence, the invention contemplates the use of charge-basedseparation to permit the separation of specific reaction products fromthe input oligonucleotides in nucleic acid-based detection assays. Thefoundation of this novel separation technique is the design and use ofoligonucleotide probes (typically termed “primers” in the case of PCR)which are “charge balanced” so that upon either cleavage or elongationof the probe it becomes “charge unbalanced,” and the specific reactionproducts may be separated from the input reactants on the basis of thenet charge.

In the context of assays which involve the elongation of anoligonucleotide probe (i.e., a primer), such as is the case in PCR, theinput primers are designed to carry a net positive charge. Elongation ofthe short oligonucleotide primer during polymerization will generate PCRproducts which now carry a net negative charge. The specific reactionproducts may then easily be separated and concentrated away from theinput primers using the charge-based separation technique describedherein (the electrodes will be reversed relative to the description inExample 24 as the product to be separated and concentrated after a PCRwill carry a negative charge).

VI. Invader™ Directed Cleavage Using Miniprobes And Mid-Range Probes

As discussed in section III above, the Invader™-directed cleavage assaymay be performed using inavder and probe oligonucleotides which have alength of about 13-25 nucleotides (typically 20-25 nucleotides). It isalso contemplated that the oligonucleotides that span the X, Y and Zregions (see FIG. 29), the invader and probe oligonucleotides, maythemselves be composed of shorter oligonucleotide sequences that alignalong a target strand but that are not covalently linked. This is to saythat there is a nick in the sugar-phosphate backbone of the compositeoligonucleotide, but that there is no disruption in the progression ofbase-paired nucleotides in the resulting duplex. When short strands ofnucleic acid align contiguously along a longer strand the hybridizationof each is stabilized by the hybridization of the neighboring fragmentsbecause the basepairs can stack along the helix as though the backbonewas in fact uninterrupted. This cooperativity of binding can give eachsegment a stability of interaction in excess of what would be expectedfor the segment hybridizing to the longer nucleic acid alone. Oneapplication of this observation has been to assemble primers for DNAsequencing, typically about 18 nucleotides long, from sets of threehexamer oligonucleotides that are designed to hybridize in this way[Kotler, L. E., et al. (1993) Proc. Natl. Acad. Sci. USA 90:4241]. Theresulting doubly-nicked primer can be extended enzymatically inreactions performed at temperatures that might be expected to disruptthe hybridization of hexamers, but not of 18-mers.

The use of composite or split oligonuceotides is applied with success inthe Invader™-directed cleavage assay. The probe oligonucleotide may besplit into two oligonucleotides which anneal in a contigious andadjacent manner along a target oligonucleotide as diagrammed in FIG. 68.In this figure, the downstream oligonucleotide (analogous to the probeof FIG. 29) is assembled from two smaller pieces: a short segment of6-10 nts (termed the “miniprobe”), that is to be cleaved in the courseof the detection reaction, and an oligonucleotide that hybridizesimmediately downstream of the miniprobe (termed the “stacker”), whichserves to stabilize the hybridization of the probe. To form the cleavagestructure, an upstream oligonucleotide (the “Invader™” oligo) isprovided to direct the cleavage activity to the desired region of theminiprobe. Assembly of the probe from non-linked pieces of nucleic acid(i.e., the miniprobe and the stacker) allows regions of sequences to bechanged without requiring the re-synthesis of the entire provensequence, thus improving the cost and flexibility of the detectionsystem. In addition, the use of unlinked composite oligonucleotidesmakes the system more stringent in its requirement of perfectly matchedhybridization to achieve signal generation, allowing this to be used asa sensitive means of detecting mutations or changes in the targetnucleic acid sequences.

As illustrated in FIG. 68, in one embodiment, the methods of the presentinvention employ at least three oligonucleotides that interact with atarget nucleic acid to form a cleavage structure for astructure-specific nuclease. More specifically, the cleavage structurecomprises i) a target nucleic acid that may be either single-stranded ordouble-stranded (when a double-stranded target nucleic acid is employed,it may be rendered single-stranded, e.g., by heating); ii) a firstoligonucleotide, termed the “stacker,” which defines a first region ofthe target nucleic acid sequence by being the complement of that region(region W of the target as shown in FIG. 67); iii) a secondoligonucleotide, termed the “miniprobe,” which defines a second regionof the target nucleic acid sequence by being the complement of thatregion (regions X and Z of the target as shown in FIG. 67); iv) a thirdoligonucleotide, termed the “invader,” the 5′ part of which defines athird region of the same target nucleic acid sequence (regions Y and Xin FIG. 67), adjacent to and downstream of the second target region(regions X and Z), and the second or 3′ part of which overlaps into theregion defined by the second oligonucleotide (region X depicts theregion of overlap). The resulting structure is diagrammed in FIG. 68.

While not limiting the invention or the instant discussion to anyparticular mechanism of action, the diagram in FIG. 68 represents theeffect on the site of cleavage caused by this type of arrangement ofthree oligonucleotides. The design of these three oligonucleotides isdescribed below in detail. In FIG. 68, the 3′ ends of the nucleic acids(i.e., the target and the oligonucleotides) are indicated by the use ofthe arrowheads on the ends of the lines depicting the strands of thenucleic acids (and where space permits, these ends are also labelled“3′”). It is readily appreciated that the three oligonucleotides (theinvader, the miniprobe and the stacker) are arranged in a parallelorientation relative to one another, while the target nucleic acidstrand is arranged in an anti-parallel orientation relative to the threeoligonucleotides. Further it is clear that the invader oligonucleotideis located upstream of the miniprobe oligonucleotide and that theminiprobe olignuceotide is located upstream of the stackeroligonucleotide and that with respect to the target nucleic acid strand,region W is upstream of region Z, region Z is upstream of upstream ofregion X and region X is upstream of region Y (that is region Y isdownstream of region X, region X is downstream of region Z and region Zis downstream of region W). Regions of complementarity between theopposing strands are indicated by the short vertical lines. While notintended to indicate the precise location of the site(s) of cleavage,the area to which the site of cleavage within the miniprobeoligonucleotide is shifted by the presence of the invaderoligonucleotide is indicated by the solid vertical arrowhead. FIG. 68 isnot intended to represent the actual mechanism of action or physicalarrangement of the cleavage structure and further it is not intendedthat the method of the present invention be limited to any particularmechanism of action.

It can be considered that the binding of these oligonucleotides dividesthe target nucleic acid into four distinct regions: one region that hascomplementarity to only the stacker (shown as “W”); one region that hascomplemetarity to only the miniprobe (shown as “Z”); one region that hascomplementarity only to the Invader™ oligo (shown as “Y”); and oneregion that has complementarity to both the Invader™ and miniprobeoligonucleotides (shown as “X”).

In addition to the benefits cited above, the use of a composite designfor the oligonucleotides which form the cleavage structure allows morelatitude in the design of the reaction conditions for performing theInvader™-directed cleavage assay. When a longer probe (e.g., 16-25 nt),as described in section III above, is used for detection in reactionsthat are performed at temperatures below the T_(m) of that probe, thecleavage of the probe may play a significant role in destabilizing theduplex of which it is a part, thus allowing turnover and reuse of therecognition site on the target nucleic acid. In contrast, withminiprobes, reaction temperatures that are at or above the T_(m) of theprobe mean that the probe molecules are hybridizing and releasing fromthe target quite rapidly even without cleavage of the probe. When anupstream Invader™ oligonucleotide and a cleavage means are provided theminiprobe will be specifically cleaved, but the cleavage will not benecessary to the turnover of the miniprobe. If a long probe (e.g., 16-25nt) were to be used in this way the temperatures required to achievethis state would be quite high, around 65 to 70° C. for a 25-mer ofaverage base composition. Requiring the use of such elevatedtemperatures limits the choice of cleavage agents to those that are verythermostable, and may contribute to background in the reactions,depending of the means of detection, through thermal degradation of theprobe oligonucleotides. Thus, the shorter probes are preferable for usein this way.

The miniprobe of the present invention may vary in size depending on thedesired application. In one embodiment, the probe may be relativelyshort compared to a standard probe (e.g., 16-25 nt), in the range of 6to 10 nucleotides. When such a short probe is used reaction conditionscan be chosen that prevent hybridization of the miniprobe in the absenceof the stacker oligonucleotide. In this way a short probe can be made toassume the statistical specificity and selectivity of a longer sequence.In the event of a perturbation in the cooperative binding of theminiprobe and stacker nucleic acids, as might be caused by a mismatchwithin the short sequence (i.e., region “Z” which is the region of theminiprobe which does not overlap with the invader) or at the junctionbetween the contiguous duplexes, this cooperativity can be lost,dramatically reducing the stability of the shorter oligonucleotide(i.e., the miniprobe), and thus reducing the level of cleaved product inthe assay of the present invention.

It is also contemplated that probes of intermediate size may be used.Such probes, in the 11 to 15 nucleotide range, may blend some of thefeatures associated with the longer probes as originally described,these features including the ability to hybridize and be cleaved absentthe help of a stacker oligonucleotide. At temperatures below theexpected T_(m) of such probes, the mechanisms of turnover may be asdiscussed above for probes in the 20 nt range, and be dependent on theremoval of the sequence in the ‘X’ region for destabilization andcycling.

The mid-range probes may also be used at elevated temperatures, at orabove their expected T_(m), to allow melting rather than cleavage topromote probe turnover. In contrast to the longer probes describedabove, however, the temperatures required to allow the use of such athermally driven turnover are much lower (about 40 to 60° C.), thuspreserving both the cleavage means and the nucleic acids in the reactionfrom thermal degradation. In this way, the mid-range probes may performin some instances like the miniprobes described above. In a furthersimilarity to the miniprobes, the accumulation of cleavage signal from amid-range probe may be helped under some reaction conditions by thepresence of a stacker.

To summarize, a standard long probe usually does not benefit from thepresence of a stacker oligonucleotide downstream (the exception beingcases where such an oligonucleotide may also disrupt structures in thetarget nucleic acid that interfere with the probe binding), and it isusually used in conditions requiring several nucleotides to be removedto allow the oligonucleotide to release from the target efficiently.

The miniprobe is very short and performs optimally in the presence of adownstream stacker oligonucleotide. The miniprobes are well suited toreactions conditions that use the temperature of the reaction to driverapid exchange of the probes on the target regardeless of whether anybases have been cleaved. In reactions with sufficient amount of thecleavage means, the probes that do bind will be rapidly cleaved beforethey melt off.

The mid-range or midiprobe combines features of these probes and can beused in reactions like those designed long probes, with longer regionsof overlap (“X” regions) to drive probe turnover at lower temperature.In a preferred embodiment, the midrange probes are used at temperaturessufficiently high that the probes are hybridizing to the target andreleasing rapidly regardless of cleavage. This is known to be thebehavior of oligonucleotides at or near their melting temperature. Thismode of turnover is more similar to that used with miniprobe/stackercombinations than with long probes. The mid-range probe may haveenhanced performance in the presence of a stacker under somecircumstances. For example, with a probe in the lower end of themid-range, e.g., 11 nt, or one with exceptional A/T content, in areaction performed well in excess of the T_(m) of the probe (e.g., >10°C. above) the presence of a stacker would be likely to enhance theperformance of the probe, while at a more moderate temperature the probemay be indifferent to a stacker.

The distinctions between the mini-, midi- (i.e., mid-range) and longprobes are not contemplated to be inflexible and based only on length.The performance of any given probe may vary with its specific sequence,the choice of solution conditions, the choice of temperature and theselected cleavage means.

It is shown in Example 18 that the assemblage of oligonucleotides thatcomprises the cleavage structure of the present invention is sensitiveto mismatches between the probe and the target. The site of the mismatchused in Ex. 18 provides one example and is not intended to be alimitation in location of a mismatch affecting cleavage. It is alsocontemplated that a mismatch between the Invader™ oligonucleotide andthe target may be used to distinguish related target sequences. In the3-oligonucleotide system, comprising an Invader™, a probe and a stackeroligonucleotide, it is contemplated that mismatches may be locatedwithin any of the regions of duplex formed between theseoligonucleotides and the target sequence. In a preferred embodiment, amismatch to be detected is located in the probe. In a particularlypreferred embodiment, the mismatch is in the probe, at the basepairimmediately upstream (i.e., 5′) of the site that is cleaved when theprobe is not mismatched to the target.

In another preferred embodiment, a mismatch to be detected is locatedwithin the region ‘Z’ defined by the hybridization of a miniprobe. In aparticularly preferred embodiment, the mismatch is in the miniprobe, atthe basepair immediately upstream (i.e., 5′) of the site that is cleavedwhen the miniprobe is not mismatched to the target.

It is also contemplated that different sequences may be detected in asingle reaction. Probes specific for the different sequences may bedifferently labeled. For example, the probes may have different dyes orother detectable moieties, different lengths, or they may havedifferences in net charges of the products after cleavage. Whendifferently labeled in one of these ways, the contribution of eachspecific target sequence to final product can be tallied. This hasapplication in detecting the quantities of different versions of a genewithin a mixture. Different genes in a mixture to be detected andquantified may be wild type and mutant genes, e.g., as may be found in atumor sample (e.g., a biopsy). In this embodiment, one might design theprobes to precisely the same site, but one to match the wild-typesequence and one to match the mutant. Quantitative detection of theproducts of cleavage from a reaction performed for a set amount of timewill reveal the ratio of the two genes in the mixture. Such analysis mayalso be performed on unrelated genes in a mixture. This type of analysisis not intended to be limited to two genes. Many variants within amixture may be similarly measured.

Alternatively, different sites on a single gene may be monitored andquantified to verify the measurement of that gene. In this embodiment,the signal from each probe would be expected to be the same.

It is also contemplated that multiple probes may be used that are notdifferently labeled, such that the aggregate signal is measured. Thismay be desirable when using many probes designed to detect a single geneto boost the signal from that gene. This configuration may also be usedfor detecting unrelated sequences within a mix. For example, in bloodbanking it is desirable to know if any one of a host of infectiousagents is present in a sample of blood. Because the blood is discardedregardless of which agent is present, different signals on the probeswould not be required in such an application of the present invention,and may actually be undesirable for reasons of confidentiality.

Just as described for the two-oligonucleotide system, above, thespecificity of the detection reaction will be influenced by theaggregate length of the target nucleic acid sequences involved in thehybridization of the complete set of the detection oligonucleotides. Forexample, there may be applications in which it is desirable to detect asingle region within a complex genome. In such a case the set ofoligonucleotides may be chosen to require accurate recognition byhybridization of a longer segment of a target nucleic acid, often in therange of 20 to 40 nucleotides. In other instances it may be desirable tohave the set of oligonucleotides interact with multiple sites within atarget sample. In these cases one approach would be to use a set ofoligonucleotides that recognize a smaller, and thus statistically morecommon, segment of target nucleic acid sequence.

In one preferred embodiment, the invader and stacker oligonucleotidesmay be designed to be maximally stable, so that they will remain boundto the target sequence for extended periods during the reaction. Thismay be accomplished through any one of a number of measures well knownto those skilled in the art, such as adding extra hybridizing sequencesto the length of the oligonucleotide (up to about 50 nts in totallength), or by using residues with reduced negative charge, such asphosphorothioates or peptide-nucleic acid residues, so that thecomplementary strands do not repel each other to degree that naturalstrands do. Such modifications may also serve to make these flankingoligonucleotides resistant to contaminating nucleases, thus furtherensuring their continued presence on the target strand during the courseof the reaction. In addition, the Invader™ and stacker oligonucleotidesmay be covalently attached to the target (e.g., through the use ofpsoralen cross-linking).

The use of the reaction temperatures at or near the T_(m) of the probeoligonucleotide, rather than the used of cleavage, to drive the turnoverof the probe oligonucleotide in these detection reactions means that theamount of the probe oligonucleotide cleaved off may be substantiallyreduced without adversely affecting the turnover rate. It has beendetermined that the relationship between the 3′ end of the upstreamoligonucleotide and the desired site of cleavage on the probe must becarefully designed. It is known that the preferred site of cleavage forthe types of structure specific endonucleases employed herein is onebasepair into a duplex (Lyamichev et al., supra). It was previouslybelieved that the presence of an upstream oligonucleotide or primerallowed the cleavage site to be shifted away from this preferred site,into the single stranded region of the 5′ arm (Lyamichev et al., supraand U.S. Pat. No. 5,422,253). In contrast to this previously proposedmechanism, and while not limiting the present invention to anyparticular mechanism, it is believed that the nucleotide immediately 5′,or upstream of the cleavage site on the probe (including miniprobe andmid-range probes) must be able to basepair with the target for efficientcleavage to occur. In the case of the present invention, this would bethe nucleotide in the probe sequence immediately upstream of theintended cleavage site. In addition, as described herein, it has beenobserved that in order to direct cleavage to that same site in theprobe, the upstream oligonucleotide must have its 3′ base (i.e., nt)immediately upstream of the the intended cleavage site of the probe.This places the 3′ terminal nucleotide of the upstream oligonucleotideand the base of the probe oligonucleotide 5′ of the cleavage site incompetition for pairing with the corresponding nucleotide of the targetstrand.

To examine the outcome of this competition, i.e. which base is pairedduring a successful cleavage event, substitutions were made in the probeand invader oligonucleotides such that either the probe or the Invader™oligonucleotide were mismatched with the target sequence at thisposition. The effects of both arrangements on the rates of cleavage wereexamined. When the Invader™ oligonucleotide is unpaired at the 3′ end,the rate of cleavage was not reduced. If this base was removed, however,the cleavage site was shifted upstream of the intended site. Incontrast, if the probe oligonucleotide was not base-paired to the targetjust upstream of the site to which the Invader™ oligonucleotide wasdirecting cleavage, the rate of cleavage was dramatically reduced,suggesting that when a competition exists, the probe oligonucleotide wasthe molecule to be base-paired in this position.

It appears that the 3′ end of the upstream invader oligonucleotide isunpaired during cleavage, and yet is required for accurate positioningof the cleavage. To examine which part(s) of the 3′ terminal nucleotideare required for the positioning of cleavage, Invader™ oligonucleotideswere designed that terminated on this end with nucleotides that werealtered in a variety of ways. Sugars examined included 2′ deoxyribosewith a 3′ phosphate group, a dideoxyribose, 3′ deoxyribose, 2′ O-methylribose, arabinose and arabinose with a 3′ phosphate. A basic ribose,with and without 3′ phosphate were tested. Synthetic “universal” basessuch at 3-nitropyrrole and 5-nitroindole on ribose sugars were tested.Finally, a base-like aromatic ring structure, acridine, linked to the 3′end the previous nucleotide without a sugar group was tested. Theresults obtained support the conclusion that the aromatic ring of thebase (at the 3′ end of the invader oligonuceotide) is the requiredmoiety for accomplishing the direction of cleavage to the desired sitewithin the downstream probe.

VII. Signal Enhancement By Tailing of Reaction Products In TheInvader™-Directed Cleavage Assay

It has been determined that when oligonucleotide probes are used incleavage detection assays at elevated temperature, some fraction of thetruncated probes will have been shortened by nonspecific thermaldegradation, and that such breakage products can make the analysis ofthe target-specific cleavage data more difficult. Background cleavagesuch as this can, when not resolved from specific cleavage products,reduce the accuracy of quantitation of target nucleic acids based on theamount of accumulated product in a set timeframe. One means ofdistinguishing the specific from the nonspecific products is disclosedabove, and is based on partitioning the products of these reactions bydifferences in the net charges carried by the different molecularspecies in the reaction. As was noted in that discussion, the thermalbreakage products usually retain 3′ phosphates after breakage, while theenzyme-cleaved products do not. The two negative charges on thephosphate facilitate charge-based partition of the products.

The absence of a 3′ phosphate on the desired subset of the probefragments may be used to advantage in enzymatic assays as well. Nucleicacid polymerases, both non-templated (e.g., terminal deoxynucleotidyltransferase, polyA polymerase) and template-dependent (e.g., Pol I-typeDNA polymerases), require an available 3′ hydroxyl by which to attachfurther nucleotides. This enzymatic selection of 3′ end structure may beused as an effective means of partitioning specific from non-specificproducts.

In addition to the benefits of the partitioning described above, theaddition of nucleotides to the end of the specific product of aninvader-specific cleavage offers an opportunity to either add label tothe products, to add capturable tails to facilitate solid-support basedreadout systems, or to do both of these things at the same time. Somepossible embodiments of this concept are illustrated in FIG. 67.

In FIG. 67, an Invader™ cleavage structure comprising an Invader™oligonuclotide containing a blocked or non-extendible 3′ end (e.g., a 3′dideoxynucleotide) and a probe oligonucleotide containing a blocked ornon-extendable 3′ end (the open circle at the 3′ end of theoligonucleotides represents a non-extendible nucleotide) and a targetnucleic acid is shown; the probe oligonucleotide may contain a 5′ endlabel such as a biotin or a fluorescein (indicated by the stars) label(cleavage structures which employ a 5′ biotin-labeled probe or a 5′fluorescein-labeled probe are shown below the large diagram of thecleavage structure to the left and the right, respectively). Following,cleavage of the probe (the site of cleavage is indicated by the largearrowhead), the cleaved biotin-labeled probe is extended using atemplate-independent polymerase (e.g., TdT) and fluoresceinatednucleotide triphosphates. The fluorescein tailed cleaved probe moleculeis then captured by silo binding via its 5′ biotin label to streptavidinand the fluroescence is then measured. Alternatively, following,cleavage of a 5′-fluoresceinated probe, the cleaved probe is extendedusing a template-independent polymerase (e.g., TdT) and dATP. Thepolyadenylated (A-tailed) cleaved probe molecule is then captured bybinding via the polyA tail to oligo dT attached to a solid support.

The examples described in FIG. 66 are based on the use of TdT to tailthe specific products of Invader™-directed cleavage. The description ofthe use of this particular enzyme is presented by way of example and isnot intended as a limitation (indeed, when probe oligos comprising RNAare employed, cleaved RNA probes may be extended using polyApolymerase). It is contemplated that an assay of this type could beconfigured to use a template-dependent polymerase, as described above.While this would require the presence of a suitable copy templatedistinct from the target nucleic acid, on which the truncatedoligonucleotide could prime synthesis, it can be envisaged that a probewhich before cleavage would be unextendible, due to either mismatch ormodification of the 3′ end, could be activated as a primer when cleavedby an invader directed cleavage. A template directed tailing reactionalso has the advantage of allowing greater selection and control of thenucleotides incorporated.

The use of nontemplated tailing does not require the presence of anyadditional nucleic acids in the detection reaction, avoiding one step ofassay development and troubleshooting. In addition, the use of nontemplated synthesis eliminated the step of hybridization, potentiallyspeeding up the assay. Furthermore, the TdT enzyme is fast, able to addat least >700 nucleotides to substrate oligonucleotides in a 15 minutereaction.

As mentioned above, the tails added can be used in a number of ways. Itcan be used as a straight-forward way of adding labeled moieties to thecleavage product to increase signal from each cleavage event. Such areaction is depicted in the left side of FIG. 66. The labeled moietiesmay be anything that can, when attached to a nucleotide, be added by thetailing enzyme, such as dye molecules, haptens such as digoxigenin, orother binding groups such as biotin.

In a preferred embodiment the assay includes a means of specificallycapturing or partitioning the tailed invader-directed cleavage productsin the mixture. It can be seen that target nucleic acids in the mixturemay be tailed during the reaction. If a label is added, it is desirableto partition the tailed invader-directed cleavage products from theseother labeled molecules to avoid background in the results. This iseasily done if only the cleavage product is capable of being captured.For example, consider a cleavage assay of the present invention in whichthe probe used has a biotin on the 5′ end and is blocked from extensionon the 3′ end, and in which a dye is added during tailing. Considerfurther that the products are to be captured onto a support via thebiotin moeity, and the captured dye measured to assess the presence ofthe target nucleic acid. When the label is added by tailing, only thespecifically cleaved probes will be labeled. The residual uncut probescan still bind in the final capture step, but they will not contributeto the signal. In the same reaction, nicks and cuts in the targetnucleic acid may be tailed by the enzyme, and thus become dye labeled.In the final capture these labeled targets will not bind to the supportand thus, though labeled, they will not contribute to the signal. If thefinal specific product is considered to consist of two portions, theprobe-derived portion and the tail portion, can be seen from thisdiscussion that it is particularly preferred that when the probe-derivedportion is used for specific capture, whether by hybridization,biotin/streptavidin, or other method, that the label be associated withthe tail portion. Conversely, if a label is attached to theprobe-derived portion, then the tail portion may be made suitable forcapture, as depicted on the right side of FIG. 66. Tails may be capturedin a number of ways, including hybridization, biotin incorporation withstreptavidin capture, or by virtue if the fact that the longer moleculesbind more predictably and efficiently to a number of nucleic acidminding matrices, such as nitrocellulose, nylon, or glass, in membrane,paper, resin, or other form. While not required for this assay, thisseparation of functions allows effective exclusion from signal of bothunreacted probe and tailed target nucleic acid.

In addition to the supports described above, the tailed products may becaptured onto any support that contains a suitable capture moiety. Forexample, biotinylated products are generally captured withavidin-treated surfaces. These avidin surfaces may be in microtitreplate wells, on beads, on dipsticks, to name just a few of thepossibilities. Such surfaces can also be modified to contain specificoligonucleotides, allowing capture of product by hybridization. Capturesurfaces as described here are generally known to those skilled in theart and include nitrocellulose dipsticks (e.g., GeneComb, BioRad,Hercules, Calif.).

VIII. Improved Enzymes For Use In Invader™-Directed Cleavage Reactions

A cleavage structure is defined herein as a structure which is formed bythe interaction of a probe oligonucleotide and a target nucleic acid toform a duplex, the resulting structure being cleavable by a cleavagemeans, including but not limited to an enzyme. The cleavage structure isfurther defined as a substrate for specific cleavage by the cleavagemeans in contrast to a nucleic acid molecule which is a substrate fornonspecific cleavage by agents such as phosphodiesterases. Examples ofsome possible cleavage structures are shown in FIG. 16. In consideringimprovements to enzymatic cleavage means, one may consider the action ofsaid enzymes on any of these structures, and on any other structuresthat fall within the definition of a cleavage structure. The cleavagesites indicated on the structures in FIG. 16 are presented by way ofexample. Specific cleavage at any site within such a structure iscontemplated.

Improvements in an enzyme may be an increased or decreased rate ofcleavage of one or more types of structures. Improvements may alsoresult in more or fewer sites of cleavage on one or more of saidcleavage structures. In developing a library of new structure-specificnucleases for use in nucleic acid cleavage assays, improvements may havemany different embodiments, each related to the specific substratestructure used in a particular assay.

As an example, one embodiment of the Invader™-directed cleavage assay ofthe present invention may be considered. In the Invader™ directedcleavage assay, the accumulation of cleaved material is influenced byseveral features of the enzyme behavior. Not surprisingly, the turnoverrate, or the number of structures that can be cleaved by a single enzymemolecule in a set amount of time, is very important in determining theamount of material processed during the course of an assay reaction. Ifan enzyme takes a long time to recognize a substrate (e.g., if it ispresented with a less-than-optimal structure), or if it takes a longtime to execute cleavage, the rate of product accumulation is lower thanif these steps proceeded quickly. If these steps are quick, yet theenzyme “holds on” to the cleaved structure, and does not immediatelyproceed to another uncut structure, the rate will be negativelyaffected.

Enzyme turnover is not the only way in which enzyme behavior cannegatively affect the rate of accumulation of product. When the meansused to visualize or measure product is specific for a precisely definedproduct, products that deviate from that definition may escapedetection, and thus the rate of product accumulation may appear to belower than it is. For example, if one had a sensitive detector fortrinucleotides that could not see di- or tetranucleotides, or any sizedoligonucleotide other that 3 residues, in the Invader™-directed cleavageassay of the present invention any errant cleavage would reduce thedetectable signal proportionally. It can be seen from the cleavage datapresented here that, while there is usually one site within a probe thatis favored for cleavage, there are often products that arise fromcleavage one or more nucleotides away from the primary cleavage site.These are products that are target dependent, and are thus notnon-specific background. Nevertheless, if a subsequent visualizationsystem can detect only the primary product, these represent a loss ofsignal. One example of such a selective visualization system is thecharge reversal readout presented herein, in which the balance ofpositive and negative charges determines the behavior of the products.In such a system the presence of an extra nucleotide or the absence ofan expected nucleotide can excluded a legitimate cleavage product fromultimate detection by leaving that product with the wrong balance ofcharge. It can be easily seen that any assay that can sensitivelydistinguish the nucleotide content of an oligonucleotide, such asstandard stringent hybridization, suffers in sensitivity when somefraction of the legitimate product is not eligible for successfuldetection by that assay.

These discussions suggest two highly desirable traits in any enzyme tobe used in the method of the present invention. First, the more rapidlythe enzyme executes an entire cleavage reaction, including recognition,cleavage and release, the more signal it may potentially created in theinvader-directed cleavage assay. Second, the more successful an enzymeis at focusing on a single cleavage site within a structure, the more ofthe cleavage product can be successfully detected in a selectiveread-out. The rationale cited above for making improvements in enzymesto be used in the Invader™-directed cleavage assay are meant to serve asan example of one direction in which improvements might be sought, butnot as a limit on either the nature or the applications of improvedenzyme activities. As another direction of activity change that would beappropriately considered improvement, the DNAP-associated 5′ nucleasesmay be used as an example. In creating some of the polymerase-deficient5′ nucleases described herein it was found that the those that werecreated by deletion of substantial portions of the polymerase domain, asdepicted in FIG. 4, assumed activities that were weak or absent in theparent proteins. These activities included the ability to cleave thenon-forked structure shown in FIG. 16D, a greatly enhanced ability toexonucleolytically remove nucleotides from the 5′ ends of duplexedstrands, and a nascent ability to cleave circular molecules withoutbenefit of a free 5′ end. These features have contributed to thedevelopment of detection assays such as the one depicted in FIG. 1A.

In addition to the 5′ nucleases derived from DNA polymerases, thepresent invention also contemplates the use of structure-specificnucleases that are not derived from DNA polymerases. For example, aclass of eukaryotic and archaebacterial endonucleases have beenidentified which have a similar substrate specificity to 5′ nucleases ofPol I-type DNA polymerases. These are the FEN1 (Flap EndoNuclease),RAD2, and XPG (Xeroderma Pigmentosa-complementation group G) proteins.These proteins are involved in DNA repair, and have been shown to favorthe cleavage of structures that resemble a 5′ arm that has beendisplaced by an extending primer during polymerization, similar to themodel depicted in FIG. 16B. Similar DNA repair enzymes have beenisolated from single cell and higher eukaryotes and from archaea, andthere are related DNA repair proteins in eubacteria. Similar 5′nucleases have also be associated with bacteriophage such as T5 and T7.

Recently, the 3-dimensional structures of DNAPTaq and T5 phage5′-exonuclease (FIG. 69) were determined by X-ray diffraction [Kim etal. (1995) Nature 376:612 and Ceska et al. (1995) Nature 382:90). Thetwo enzymes have very similar 3-dimensional structures despite limitedamino acid sequence similarity. The most striking feature of the T55′-exonuclease structure is the existence of a triangular hole formed bythe active site of the protein and two alpha helices (FIG. 69). Thissame region of DNAPTaq is disordered in the crystal structure,indicating that this region is flexible, and thus is not shown in thepublished 3-dimensional structure. However, the 5′ nuclease domain ofDNAPTaq is likely to have the same structure, based its overall3-dimensional similarity to T5 5′-exonuclease, and that the amino acidsin the disordered region of the DNAPTaq protein are those associatedwith alpha helix formation. The existence of such a hole or groove inthe 5′ nuclease domain of DNAPTaq was predicted based on its substratespecificity [Lyamichev et al., supra].

It has been suggested that the 5′ arm of a cleavage structure mustthread through the helical arch described above to position saidstructure correctly for cleavage (Ceska et al., supra). One of themodifications of 5′ nucleases described herein opened up the helicalarch portion of the protein to allow improved cleavage of structuresthat cut poorly or not at all (e.g., structures on circular DNA targetsthat would preclude such threading of a 5′ arm). The gene construct thatwas chosen as a model to test this approach was the one called Cleavase®BN, which was derived from DNAPTaq but does not contain the polymerasedomainn (Ex. 2). It comprises the entire 5′ nuclease domain of DNAP Taq,and thus should be very close in structure to the T5 5′ exonuclease.This 5′ nuclease was chosen to demonstrate the principle of such aphysical modification on proteins of this type. The arch-openingmodification of the present invention is not intended to be limited tothe 5′ nuclease domains of DNA polymerases, and is contemplated for useon any structure-specific nuclease which includes such an aperture as alimitation on cleavage activity.

The opening of the helical arch was accomplished by insertion of aprotease site in the arch. This allowed post-translational digestion ofthe expressed protein with the appropriate protease to open the arch atits apex. Proteases of this type recognize short stretches of specificamino acid sequence. Such proteases include thrombin and factor Xa.Cleavage of a protein with such a protease depends on both the presenceof that site in the amino acid sequence of the protein and theaccessibility of that site on the folded intact protein. Even with acrystal structure it can be difficult to predict the susceptibility ofany particular region of a protein to protease cleavage. Absent acrystal structure it must be determined empirically.

In selecting a protease for a site-specific cleavage of a protein thathas been modified to contain a protease cleavage site, a first step isto test the unmodified protein for cleavage at alternative sites. Forexample, DNAPTaq and Cleavase® BN nuclease were both incubated underprotease cleavage conditions with factor Xa and thrombin proteases. Bothnuclease proteins were cut with factor Xa within the 5′ nuclease domain,but neither nuclease was digested with large amounts of thrombin. Thus,thrombin was chosen for initial tests on opening the arch of theCleavase® BN enzyme.

In the protease/Cleavaseg modifications described herein the factor Xaprotease cleaved strongly in an unacceptable position in the unmodifiednuclease protein, in a region likely to compromise the activity of theend product. Other unmodified nucleases contemplated herein may not besensitive to the factor Xa, but may be sensitive to thrombin or othersuch proteases. Alternatively, they may be sensitive to these or othersuch proteases at sites that are immaterial to the function of thenuclease sought to be modified. In approaching any protein formodification by addition of a protease cleavage site, the unmodifiedprotein should be tested with the proteases under consideration todetermine which proteases give acceptable levels of cleavage in otherregions.

Working with the cloned segment of DNAPTaq from which the Cleavase® BNprotein is expressed, nucleotides encoding a thrombin cleavage site wereintroduced in-frame near the sequence encoding amino acid 90 of thenuclease gene. This position was determined to be at or near the apex ofthe helical arch by reference to both the 3-dimensional structure ofDNAPTaq, and the structure of T5 5′ exonuclease.

The encoded amino acid sequence, LVPRGS, was inserted into the apex ofthe helical arch by site-directed mutagenesis of the nuclease gene. Theproline (P) in the thrombin cleavage site was positioned to replace aproline normally in this position in Cleavase® BN because proline is analpha helix-breaking amino acid, and may be important for the3-dimensional structure of this arch. This construct was expressed,purified and then digested with thrombin. The digested enzyme was testedfor its ability to cleave a target nucleic acid, bacteriophage M13genomic DNA, that does not provide free 5′ ends to facilitate cleavageby the threading model.

While the helical arch in this nuclease was opened by protease cleavage,it is contemplated that a number of other techniques could be used toachieve the same end. For example, the nucleotide sequence could berearranged such that, upon expression, the resulting protein would beconfigured so that the top of the helical arch (amino acid 90) would beat the amino terminus of the protein, the natural carboxyl and aminotermini of the protein sequence would be joined, and the new carboxylterminus would lie at natural amino acid 89. This approach has thebenefit that no foreign sequences are introduced and the enzyme is asingle amino acid chain, and thus may be more stable that the cleaved 5′nuclease. In the crystal structure of DNAPTaq, the amino and carboxyltermini of the 5′-exonuclease domain lie in close proximity to eachother, which suggests that the ends may be directly joined without theuse of a flexible linker peptide sequence as is sometimes necessary.Such a rearrangement of the gene, with subsequent cloning and expressioncould be accomplished by standard PCR recombination and cloningtechniques known to those skilled in the art.

The present invention also contemplates the use of nucleases isolatedfrom a organisms that grow under a variety of conditions. The genes forthe FEN-1/XPG class of enzymes are found in organisms ranging frombacteriophage to humans to the extreme thermophiles of Kingdom Archaea.For assays in which high temperature is to be used, it is contemplatedthat enzymes isolated from extreme thermophiles may exhibit thethermostability required of such an assay. For assays in which it mightbe desirable to have peak enzyme activity at moderate temperature or inwhich it might be desirable to destroy the enzyme with elevatedtemperature, those enzymes from organisms that favor moderatetemperatures for growth may be of particular value.

An alignment of a collection of FEN-1 proteins sequenced by others isshown in FIGS. 70A-E. It can be seen from this alignment that there aresome regions of conservation in this class of proteins, suggesting thatthey are related in function, and possibly in structure. Regions ofsimilarity at the amino acid sequence level can be used to designprimers for in vitro amplification (PCR) by a process of backtranslating the amino acid sequence to the possible nucleic acidsequences, then choosing primers with the fewest possible variationswithin the sequences. These can be used in low stringency PCR to searchfor related DNA sequences. This approach permits the amplification ofDNA encoding a FEN-1 nuclease without advance knowledge of the actualDNA sequence.

It can also be seen from this alignment that there are regions in thesequences that are not completely conserved. The degree of differenceobserved suggests that the proteins may have subtle or distinctdifferences is substrate specificity. In other words, they may havedifferent levels of cleavage activity on the cleavage structures of thepresent invention. When a particular structure is cleaved at a higherrate than the others, this is referred to a preferred substrate, while astructure that is cleaved slowly is considered a less preferredsubstrate. The designation of preferred or less preferred substrates inthis context is not intended to be a limitation of the presentinvention. It is contemplated that some embodiments the presentinvention will make use of the interactions of an enzyme with a lesspreferred substrate. Candidate enzymes are tested for suitability in thecleavage assays of the present invention using the assays describedbelow.

-   -   1. Structure Specific Nuclease Assay

Testing candidate nucleases for structure-specific activities in theseassays is done in much the same way as described for testing modifiedDNA polymerases in Example 2, but with the use of a different library ofmodel structures. In addition to assessing the enzyme performance inprimer-independent and primer-directed cleavage, a set of synthetichairpins are used to examine the length of duplex downstream of thecleavage site preferred by the enzyme.

The FEN-1 and XPG 5′ nucleases used in the present invention must betested for activity in the assays in which they are intended to be used,including but not limited to the Invader™-directed cleavage detectionassay of the present invention and the CFLP® method of characterizingnucleic acids (the CFLP® method is described in co-pending applicationSer. Nos. 08/337,164, 08/402,601, 08/484,956 and 08/520,946; thedisclosures of these applications are incorporated herein by reference).The Invader™ assay uses a mode of cleavage that has been termed “primerdirected” of “primer dependent” to reflect the influence of the anoligonucleotide hybridized to the target nucleic acid upstream of thecleavage site. In contrast, the CFLP® reaction is based on the cleavageof folded structure, or hairpins, within the target nucleic acid, in theabsence of any hybridized oligonucleotide. The tests described hereinare not intended to be limited to the analysis of nucleases with anyparticular site of cleavage or mode of recognition of substratestructures. It is contemplated that enzymes may be described as 3′nucleases, utilizing the 3′ end as a reference point to recognizestructures, or may have a yet a different mode of recognition. Further,the use of the term 5′ nucleases is not intended to limit considerationto enzymes that cleave the cleavage structures at any particular site.It refers to a general class of enzymes that require some reference oraccess to a 5′ end to effect cleavage of a structure.

A set of model cleavage structures have been created to allow thecleavage ability of unknown enzymes on such structures to be assessed.Each of the model structures is constructed of one or more syntheticoligonucleotides made by standard DNA synthesis chemistry. Examples ofsuch synthetic model substrate structures are shown in FIGS. 30 and 70.These are intended only to represent the general folded configurationdesirable is such test structures. While a sequence that would assumesuch a structure is indicated in the figures, there are numerous othersequence arrangements of nucleotides that would be expected to fold insuch ways. The essential features to be designed into a set ofoligonucleotides to perform the tests described herein are the presenceor absence of a sufficiently long 3′ arm to allow hybridization of anadditional nucleic acid to test cleavage in a “primer-directed” mode,and the length of the duplex region. In the set depicted in FIG. 71, theduplex lengths of the S-33 and the 11-8-0 structures are 12 and 8basepairs, respectively. This difference in length in the test moleculesfacilitates detection of discrimination by the candidate nucleasebetween longer and shorter duplexes. Additions to this series expandingthe range of duplex molecules presented to the enzymes, both shorter andlonger, may be used. The use of a stabilizing DNA tetraloop [Antao etal. (1991) Nucl. Acids Res. 19:5901] or triloop [Hiraro et al. (1994)Nuc. Acids Res. 22:576] at the closed end of the duplex helps ensureformation of the expected structure by the oligonucleotide.

The model substrate for testing primer directed cleavage, the “S-60hairpin” (SEQ ID NO:40) is described in Example 11. In the absence of aprimer this hairpin is usually cleaved to release 5′ arm fragments of 18and 19 nucleotides length. An oligonucleotide, termed P-14(5′-CGAGAGACCACGCT-3′), that extends to the base of the duplex whenhybridized to the 3′ arm of the S-60 hairpin gives cleavage products ofthe same size, but at a higher rate of cleavage.

To test invasive cleavage a different primer is used, termed P-15(5′-CGAGAGACCACGCTG -3′). In a successful invasive cleavage the presenceof this primer shifts the site of cleavage of S-60 into the duplexregion, usually releasing products of 21 and 22 nucleotides length.

The S-60 hairpin may also be used to test the effects of modificationsof the cleavage structure on either primer-directed or invasivecleavage. Such modifications include, but are not limited to, use ofmismatches or base analogs in the hairpin duplex at one, a few or allpositions, similar disruptions or modifications in the duplex betweenthe primer and the 3′ arm of the S-60, chemical or other modificationsto one or both ends of the primer sequence, or attachment of moietiesto, or other modifications of the 5′ arm of the structure. In all of theanalyses using the S-60 or a similar hairpin described herein, activitywith and without a primer may be compared using the same hairpinstructure.

The assembly of these test reactions, including appropriate amounts ofhairpin, primer and candidate nuclease are described in Example 2. Ascited therein, the presence of cleavage products is indicated by thepresence of molecules which migrate at a lower molecular weight thandoes the uncleaved test structure. When the reversal of charge of alabel is used the products will carry a different net charge than theuncleaved material. Any of these cleavage products indicate that thecandidate nuclease has the desired structure-specific nuclease activity.By “desired structure-specific nuclease activity” it is meant only thatthe candidate nuclease cleaves one or more test molecules. It is notnecessary that the candidate nuclease cleave at any particular rate orsite of cleavage to be considered successful cleavage.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the disclosure which follows, the following abbreviations apply:° C.(degrees Centigrade); g (gravitational field); vol (volume); w/v (weightto volume); v/v (volume to volume); BSA (bovine serum albumin); CTAB(cetyltrimethylammonium bromide); HPLC (high pressure liquidchromatography); DNA (deoxyribonucleic acid); p (plasmid); μl(microliters); ml (milliliters); μg (micrograms); pmoles (picomoles); mg(milligrams); M (molar); mM (milliMolar); μM (microMolar); nm(nanometers); kdal (kilodaltons); OD (optical density); EDTA (ethylenediamine tetra-acetic acid); FITC (fluorescein isothiocyanate); SDS(sodium dodecyl sulfate); NaPO₄ (sodium phosphate); Tris(tris(hydroxymethyl)-aminomethane); PMSF (phenyhnethylsulfonylfluoride);TBE (Tris-Borate-EDTA, i.e., Tris buffer titrated with boric acid ratherthan HCl and containing EDTA) ; PBS (phosphate buffered saline); PPBS(phosphate buffered saline containing 1 mM PMSF); PAGE (polyacrylamidegel electrophoresis); Tween (polyoxyethylene-sorbitan); Dynal (Dynal A.S., Oslo, Norway); Epicentre (Epicentre Technologies, Madison, Wis.); MJResearch (MJ Research, Watertown, Mass.); National Biosciences(Plymouth, Minn.); New England Biolabs (Beverly, Mass.); Novagen(Novagen, Inc., Madison, Wis.); Perkin Elmer (Norwalk, Conn.); PromegaCorp. (Madison, Wis.); Stratagene (Stratagene Cloning Systems, La Jolla,Calif.); USB (U.S. Biochemical, Cleveland, Ohio).

EXAMPLE 1 Characteristics of Native Thermostable DNA Polymerases

A. 5′ Nuclease Activity of DNAPTaq

During the polymerase chain reaction (PCR) [Saiki et al., Science239:487 (1988); Mullis and Faloona, Methods in Enzymology 155:335(1987)], DNAPTaq is able to amplify many, but not all, DNA sequences.One sequence that cannot be amplified using DNAPTaq is shown in FIG. 6(Hairpin structure is SEQ ID NO:15, PRIMERS are SEQ ID NOS:16-17.) ThisDNA sequence has the distinguishing characteristic of being able to foldon itself to form a hairpin with two single-stranded arms, whichcorrespond to the primers used in PCR.

To test whether this failure to amplify is due to the 5′ nucleaseactivity of the enzyme, we compared the abilities of DNAPTaq and DNAPStfto amplify this DNA sequence during 30 cycles of PCR. Syntheticoligonucleotides were obtained from The Biotechnology Center at theUniversity of Wisconsin-Madison. The DNAPTaq and DNAPStf were fromPerkin Elmer (i.e., Amplitaq™ DNA polymerase and the Stoffel fragment ofAmplitaq™ DNA polymerase). The substrate DNA comprised the hairpinstructure shown in FIG. 6 cloned in a double-stranded form into pUC19.The primers used in the amplification are listed as SEQ ID NOS:16-17.Primer SEQ ID NO:17 is shown annealed to the 3′ arm of the hairpinstructure in FIG. 6. Primer SEQ ID NO:16 is shown as the first 20nucleotides in bold on the 5′ arm of the hairpin in FIG. 6.

Polymerase chain reactions comprised 1 ng of supercoiled plasmid targetDNA, 5 pmoles of each primer, 40 μM each dNTP, and 2.5 units of DNAPTaqor DNAPStf, in a 50 μl solution of 10 mM Tris•Cl pH 8.3. The DNAPTaqreactions included 50 mM KCl and 1.5 mM MgCl₂. The temperature profilewas 95° C. for 30 sec., 55° C. for 1 min. and 72° C. for 1 min., through30 cycles. Ten percent of each reaction was analyzed by gelelectrophoresis through 6% polyacrylamide (cross-linked 29:1) in abuffer of 45 mM Tris•Borate, pH 8.3, 1.4 mM EDTA.

The results are shown in FIG. 7. The expected product was made byDNAPStf (indicated simply as “S”) but not by DNAPTaq (indicated as “T”).We conclude that the 5′ nuclease activity of DNAPTaq is responsible forthe lack of amplification of this DNA sequence.

To test whether the 5′ unpaired nucleotides in the substrate region ofthis its structured DNA are removed by DNAPTaq, the fate of theend-labeled 5′ arm during four cycles of PCR was compared using the sametwo polymerases (FIG. 8). The hairpin templates, such as the onedescribed in FIG. 6, were made using DNAPStf and a ³²P-5′-end-labeledprimer. The 5′-end of the DNA was released as a few large fragments byDNAPTaq but not by DNAPStf. The sizes of these fragments (based on theirmobilities) show that they contain most or all of the unpaired 5′ arm ofthe DNA. Thus, cleavage occurs at or near the base of the bifurcatedduplex. These released fragments terminate with 3′ OH groups, asevidenced by direct sequence analysis, and the abilities of thefragments to be extended by terminal deoxynucleotidyl transferase.

FIGS. 9-11 show the results of experiments designed to characterize thecleavage reaction catalyzed by DNAPTaq. Unless otherwise specified, thecleavage reactions comprised 0.01 pmoles of heat-denatured, end-labeledhairpin DNA (with the unlabeled complementary strand also present), 1pmole primer (complementary to the 3′ arm) and 0.5 units of DNAPTaq(estimated to be 0.026 pmoles) in a total volume of 10 μl of 10 mMTris-Cl, ph 8.5, 50 mM KCl and 1.5 mM MgCl₂. As indicated, somereactions had different concentrations of KCl, and the precise times andtemperatures used in each experiment are indicated in the individualfigures. The reactions that included a primer used the one shown in FIG.6 (SEQ ID NO:17). In some instances, the primer was extended to thejunction site by providing polymerase and selected nucleotides.

Reactions were initiated at the final reaction temperature by theaddition of either the MgCl₂ or enzyme. Reactions were stopped at theirincubation temperatures by the addition of 8 μl of 95% formamide with 20mM EDTA and 0.05% marker dyes. The T_(m) calculations listed were madeusing the Oligo™ primer analysis software from National Biosciences,Inc. These were determined using 0.25 μM as the DNA concentration, ateither 15 or 65 mM total salt (the 1.5 mM MgCl₂ in all reactions wasgiven the value of 15 mM salt for these calculations).

FIG. 9 is an autoradiogram containing the results of a set ofexperiments and conditions on the cleavage site. FIG. 9A is adetermination of reaction components that enable cleavage. Incubation of5′-end-labeled hairpin DNA was for 30 minutes at 55° C., with theindicated components. The products were resolved by denaturingpolyacrylamide gel electrophoresis and the lengths of the products, innucleotides, are indicated. FIG. 9B describes the effect of temperatureon the site of cleavage in the absence of added primer. Reactions wereincubated in the absence of KCl for 10 minutes at the indicatedtemperatures. The lengths of the products, in nucleotides, areindicated.

Surprisingly, cleavage by DNAPTaq requires neither a primer nor dNTPs(see FIG. 9A). Thus, the 5′ nuclease activity can be uncoupled frompolymerization. Nuclease activity requires magnesium ions, thoughmanganese ions can be substituted, albeit with potential changes inspecificity and activity. Neither zinc nor calcium ions support thecleavage reaction. The reaction occurs over a broad temperature range,from 25° C. to 85° C., with the rate of cleavage increasing at highertemperatures.

Still referring to FIG. 9, the primer is not elongated in the absence ofadded dNTPs. However, the primer influences both the site and the rateof cleavage of the hairpin. The change in the site of cleavage (FIG. 9A)apparently results from disruption of a short duplex formed between thearms of the DNA substrate. In the absence of primer, the sequencesindicated by underlining in FIG. 6 could pair, forming an extendedduplex. Cleavage at the end of the extended duplex would release the 11nucleotide fragment seen on the FIG. 9A lanes with no added primer.Addition of excess primer (FIG. 9A, lanes 3 and 4) or incubation at anelevated temperature (FIG. 9B) disrupts the short extension of theduplex and results in a longer 5′ arm and, hence, longer cleavageproducts.

The location of the 3′ end of the primer can influence the precise siteof cleavage. Electrophoretic analysis revealed that in the absence ofprimer (FIG. 9B), cleavage occurs at the end of the substrate duplex(either the extended or shortened form, depending on the temperature)between the first and second base pairs. When the primer extends up tothe base of the duplex, cleavage also occurs one nucleotide into theduplex. However, when a gap of four or six nucleotides exists betweenthe 3′ end of the primer and the substrate duplex, the cleavage site isshifted four to six nucleotides in the 5′ direction.

FIG. 10 describes the kinetics of cleavage in the presence (FIG. 10A) orabsence (FIG. 10B) of a primer oligonucleotide. The reactions were runat 55° C. with either 50 mM KCl (FIG. 10A) or 20 mM KCl (FIG. 10B). Thereaction products were resolved by denaturing polyacrylamide gelelectrophoresis and the lengths of the products, in nucleotides, areindicated. “M”, indicating a marker, is a 5′ end-labeled 19-ntoligonucleotide. Under these salt conditions, FIGS. 10A and 10B indicatethat the reaction appears to be about twenty times faster in thepresence of primer than in the absence of primer. This effect on theefficiency may be attributable to proper alignment and stabilization ofthe enzyme on the substrate.

The relative influence of primer on cleavage rates becomes much greaterwhen both reactions are run in 50 mM KCl. In the presence of primer, therate of cleavage increases with KCl concentration, up to about 50 mM.However, inhibition of this reaction in the presence of primer isapparent at 100 mM and is complete at 150 mM KCl. In contrast, in theabsence of primer the rate is enhanced by concentration of KCl up to 20mM, but it is reduced at concentrations above 30 mM. At 50 mM KCl, thereaction is almost completely inhibited. The inhibition of cleavage byKCl in the absence of primer is affected by temperature, being morepronounced at lower temperatures.

Recognition of the 5′ end of the arm to be cut appears to be animportant feature of substrate recognition. Substrates that lack a free5′ end, such as circular M13 DNA, cannot be cleaved under any conditionstested. Even with substrates having defined 5′ arms, the rate ofcleavage by DNAPTaq is influenced by the length of the arm. In thepresence of primer and 50 mM KCl, cleavage of a 5′ extension that is 27nucleotides long is essentially complete within 2 minutes at 55° C. Incontrast, cleavages of molecules with 5′ arms of 84 and 188 nucleotidesare only about 90% and 40% complete after 20 minutes. Incubation athigher temperatures reduces the inhibitory effects of long extensionsindicating that secondary structure in the 5′ arm or a heat-labilestructure in the enzyme may inhibit the reaction. A mixing experiment,run under conditions of substrate excess, shows that the molecules withlong arms do not preferentially tie up the available enzyme innon-productive complexes. These results may indicate that the 5′nuclease domain gains access to the cleavage site at the end of thebifurcated duplex by moving down the 5′ arm from one end to the other.Longer 5′ arms would be expected to have more adventitious secondarystructures (particularly when KCl concentrations are high), which wouldbe likely to impede this movement.

Cleavage does not appear to be inhibited by long 3′ arms of either thesubstrate strand target molecule or pilot nucleic acid, at least up to 2kilobases. At the other extreme, 3′ arms of the pilot nucleic acid asshort as one nucleotide can support cleavage in a primer-independentreaction, albeit inefficiently. Fully paired oligonucleotides do notelicit cleavage of DNA templates during primer extension.

The ability of DNAPTaq to cleave molecules even when the complementarystrand contains only one unpaired 3′ nucleotide may be useful inoptimizing allele-specific PCR. PCR primers that have unpaired 3′ endscould act as pilot oligonucleotides to direct selective cleavage ofunwanted templates during preincubation of potential template-primercomplexes with DNAPTaq in the absence of nucleoside triphosphates.

B. 5′ Nuclease Activities of Other DNAPs

To determine whether other 5′ nucleases in other DNAPs would be suitablefor the present invention, an array of enzymes, several of which werereported in the literature to be free of apparent 5′ nuclease activity,were examined. The ability of these other enzymes to cleave nucleicacids in a structure-specific manner was tested using the hairpinsubstrate shown in FIG. 6 under conditions reported to be optimal forsynthesis by each enzyme.

DNAPEc1 and DNAP Klenow were obtained from Promega Corporation; the DNAPof Pyrococcus furious [“Pfu”, Bargseid et al., Strategies 4:34 (1991)]was from Strategene; the DNAP of Thermococcus litoralis [“Tli”,Vent™(exo-), Perler et al., Proc. Natl. Acad. Sci. USA 89:5577 (1992)]was from New England Biolabs; the DNAP of Thermus flavus [“Tfl”, Kaledinet al., Biokhimiya 46:1576 (1981)] was from Epicentre Technologies; andthe DNAP of Thermus thermophilus [“Tth”, Carballeira et al.,Biotechniques 9:276 (1990); Myers et al., Biochem. 30:7661 (1991)] wasfrom U.S. Biochemicals.

0.5 units of each DNA polymerase was assayed in a 20 μl reaction, usingeither the buffers supplied by the manufacturers for theprimer-dependent reactions, or 10 mM Tris•Cl, pH 8.5, 1.5 mM MgCl₂, and20 mM KCl. Reaction mixtures were at held 72° C. before the addition ofenzyme.

FIG. 11 is an autoradiogram recording the results of these tests. FIG.11A demonstrates reactions of endonucleases of DNAPs of severalthermophilic bacteria. The reactions were incubated at 55° C. for 10minutes in the presence of primer or at 72° C. for 30 minutes in theabsence of primer, and the products were resolved by denaturingpolyacrylamide gel electrophoresis. The lengths of the products, innucleotides, are indicated. FIG. 11B demonstrates endonucleolyticcleavage by the 5′ nuclease of DNAPEc1. The DNAPEc1 and DNAP Klenowreactions were incubated for 5 minutes at 37° C. Note the light band ofcleavage products of 25 and 11 nucleotides in the DNAPEc1 lanes (made inthe presence and absence of primer, respectively). FIG. 7B alsodemonstrates DNAPTaq reactions in the presence (+) or absence (−) ofprimer. These reactions were run in 50 mM and 20 mM KCl, respectively,and were incubated at 55° C. for 10 minutes.

Referring to FIG. 11A, DNAPs from the eubacteria Thermus thermophilusand Thermus flavus cleave the substrate at the same place as DNAPTaq,both in the presence and absence of primer. In contrast, DNAPs from thearchaebacteria Pyrococcus furiosus and Thermococcus litoralis are unableto cleave the substrates endonucleolytically. The DNAPs from Pyrococcusfurious and Thermococcus litoralis share little sequence homology witheubacterial enzymes (Ito et al., Nucl. Acids Res. 19:4045 (1991); Mathuret al., Nucl. Acids. Res. 19:6952 (1991); see also Perler et al.).Referring to FIG. 11B, DNAPEc1 also cleaves the substrate, but theresulting cleavage products are difficult to detect unless the 3′exonuclease is inhibited. The amino acid sequences of the 5′ nucleasedomains of DNAPEc1 and DNAPTaq are about 38% homologous (Gelfand,supra).

The 5′ nuclease domain of DNAPTaq also shares about 19% homology withthe 5′ exonuclease encoded by gene 6 of bacteriophage T7 [Dunn et al.,J. Mol. Biol. 166:477 (1983)]. This nuclease, which is not covalentlyattached to a DNAP polymerization domain, is also able to cleave DNAendonucleolytically, at a site similar or identical to the site that iscut by the 5′ nucleases described above, in the absence of addedprimers.

C. Transcleavage

The ability of a 5′ nuclease to be directed to cleave efficiently at anyspecific sequence was demonstrated in the following experiment. Apartially complementary oligonucleotide termed a “pilot oligonucleotide”was hybridized to sequences at the desired point of cleavage. Thenon-complementary part of the pilot oligonucleotide provided a structureanalogous to the 3′ arm of the template (see FIG. 6), whereas the 5′region of the substrate strand became the 5′ arm. A primer was providedby designing the 3′ region of the pilot so that it would fold on itselfcreating a short hairpin with a stabilizing tetra-loop [Antao et al.,Nucl. Acids Res. 19:5901 (1991)]. Two pilot oligonucleotides are shownin FIG. 12A. Oligonucleotides 19-12 (SEQ ID NO:18), 30-12 (SEQ ID NO:19)and 30-0 (SEQ ID NO:20) are 31, 42 or 30 nucleotides long, respectively.However, oligonucleotides 19-12 (SEQ ID NO:18) and 34-19 (SEQ ID NO:19)have only 19 and 30 nucleotides, respectively, that are complementary todifferent sequences in the substrate strand. The pilot oligonucleotidesare calculated to melt off their complements at about 50° C. (19-12) andabout 75° C. (30-12). Both pilots have 12 nucleotides at their 3′ ends,which act as 3′ arms with base-paired primers attached.

To demonstrate that cleavage could be directed by a pilotoligonucleotide, we incubated a single-stranded target DNA with DNAPTaqin the presence of two potential pilot oligonucleotides. Thetranscleavage reactions, where the target and pilot nucleic acids arenot covalently linked, includes 0.01 pmoles of single end-labeledsubstrate DNA, 1 unit of DNAPTaq and 5 pmoles of pilot oligonucleotidein a volume of 20 μl of the same buffers. These components were combinedduring a one minute incubation at 95° C., to denature the PCR-generateddouble-stranded substrate DNA, and the temperatures of the reactionswere then reduced to their final incubation temperatures.Oligonucleotides 30-12 and 19-12 can hybridize to regions of thesubstrate DNAs that are 85 and 27 nucleotides from the 5′ end of thetargeted strand.

FIG. 21 shows the complete 206-mer sequence (SEQ ID NO:32). The 206-merwas generated by PCR. The M13/pUC 24-mer reverse sequencing (−48) primerand the M13/pUC sequencing (−47) primer from New England Biolabs(catalogue nos. 1233 and 1224 respectively) were used (50 pmoles each)with the pGEM3z(f+) plasmid vector (Promega Corp.) as template (10 ng)containing the target sequences. The conditions for PCR were as follows:50 μM of each dNTP and 2.5 units of Taq DNA polymerase in 100 μl of 20mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl with 0.05% Tween-20 and0.05% NP-40. Reactions were cycled 35 times through 95° C. for 45seconds, 63° C. for 45 seconds, then 72° C. for 75 seconds. Aftercycling, reactions were finished off with an incubation at 72° C. for 5minutes. The resulting fragment was purified by electrophoresis througha 6% polyacrylamide gel (29:1 cross link) in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA, visualized by ethidium bromidestaining or autoradiography, excised from the gel, eluted by passivediffusion, and concentrated by ethanol precipitation.

Cleavage of the substrate DNA occurred in the presence of the pilotoligonucleotide 19-12 at 50° C. (FIG. 12B, lanes 1 and 7) but not at 75°C. (lanes 4 and 10). In the presence of oligonucleotide 30-12 cleavagewas observed at both temperatures. Cleavage did not occur in the absenceof added oligonucleotides (lanes 3, 6 and 12) or at about 80° C. eventhough at 50° C. adventitious structures in the substrate allowedprimer-independent cleavage in the absence of KCl (FIG. 12B, lane 9). Anon-specific oligonucleotide with no complementarity to the substrateDNA did not direct cleavage at 50° C., either in the absence or presenceof 50 mM KCl (lanes 13 and 14). Thus, the specificity of the cleavagereactions can be controlled by the extent of complementarity to thesubstrate and by the conditions of incubation.

D. Cleavage of RNA

An shortened RNA version of the sequence used in the transcleavageexperiments discussed above was tested for its ability to serve as asubstrate in the reaction. The RNA is cleaved at the expected place, ina reaction that is dependent upon the presence of the pilotoligonucleotide. The RNA substrate, made by T7 RNA polymerase in thepresence of [α-³²P]UTP, corresponds to a truncated version of the DNAsubstrate used in FIG. 12B. Reaction conditions were similar to those inused for the DNA substrates described above, with 50 mM KCl; incubationwas for 40 minutes at 55° C. The pilot oligonucleotide used is termed30-0 (SEQ ID NO:20) and is shown in FIG. 13A.

The results of the cleavage reaction is shown in FIG. 13B. The reactionwas run either in the presence or absence of DNAPTaq or pilotoligonucleotide as indicated in FIG. 13B.

Strikingly, in the case of RNA cleavage, a 3′ arm is not required forthe pilot oligonucleotide. It is very unlikely that this cleavage is dueto previously described RNaseH, which would be expected to cut the RNAin several places along the 30 base-pair long RNA-DNA duplex. The 5′nuclease of DNAPTaq is a structure-specific RNaseH that cleaves the RNAat a single site near the 5′ end of the heteroduplexed region.

It is surprising that an oligonucleotide lacking a 3′ arm is able to actas a pilot in directing efficient cleavage of an RNA target because sucholigonucleotides are unable to direct efficient cleavage of DNA targetsusing native DNAPs. However, some 5′ nucleases of the present invention(for example, clones E, F and G of FIG. 4) can cleave DNA in the absenceof a 3′ arm. In other words, a non-extendable cleavage structure is notrequired for specific cleavage with some 5′ nucleases of the presentinvention derived from thermostable DNA polymerases.

We tested whether cleavage of an RNA template by DNAPTaq in the presenceof a fully complementary primer could help explain why DNAPTaq is unableto extend a DNA oligonucleotide on an RNA template, in a reactionresembling that of reverse transcriptase. Another thermophilic DNAP,DNAPTth, is able to use RNA as a template, but only in the presence ofMn++, so we predicted that this enzyme would not cleave RNA in thepresence of this cation. Accordingly, we incubated an RNA molecule withan appropriate pilot oligonucleotide in the presence of DNAPTaq orDNAPTth, in buffer containing either Mg++ or Mn++. As expected, bothenzymes cleaved the RNA in the presence of Mg++. However, DNAPTaq, butnot DNAPTth, degraded the RNA in the presence of Mni++. We conclude thatthe 5′ nuclease activities of many DNAPs may contribute to theirinability to use RNA as templates.

EXAMPLE 2 Generation of 5′ Nucleases From Thermostable DNA Polymerases

Thermostable DNA polymerases were generated which have reduced syntheticactivity, an activity that is an undesirable side-reaction during DNAcleavage in the detection assay of the invention, yet have maintainedthermostable nuclease activity. The result is a thermostable polymerasewhich cleaves nucleic acids DNA with extreme specificity.

Type A DNA polymerases from eubacteria of the genus Thermus shareextensive protein sequence identity (90% in the polymerization domain,using the Lipman-Pearson method in the DNA analysis software fromDNAStar, Wis.) and behave similarly in both polymerization and nucleaseassays. Therefore, we have used the genes for the DNA polymerase ofThermus aquaticus (DNAPTaq) and Thermus flavus (DNAPTfl) asrepresentatives of this class. Polymerase genes from other eubacterialorganisms, such as Thermus thermophilus, Thermus sp., Thermotogamaritima, Thermosipho africanus and Bacillus stearothermophilus areequally suitable. The DNA polymerases from these thermophilic organismsare capable of surviving and performing at elevated temperatures, andcan thus be used in reactions in which temperature is used as aselection against non-specific hybridization of nucleic acid strands.

The restriction sites used for deletion mutagenesis, described below,were chosen for convenience. Different sites situated with similarconvenience are available in the Thermus thermophilus gene and can beused to make similar constructs with other Type A polymerase genes fromrelated organisms.

A. Creation of 5′ Nuclease Constructs

-   -   1. Modified DNAPTaq Genes

The first step was to place a modified gene for the Taq DNA polymeraseon a plasmid under control of an inducible promoter. The modified Taqpolymerase gene was isolated as follows: The Taq DNA polymerase gene wasamplified by polymerase chain reaction from genomic DNA from Thermusaquaticus, strain YT-1 (Lawyer et al., supra), using as primers theoligonucleotides described in SEQ ID NOS:13-14. The resulting fragmentof DNA has a recognition sequence for the restriction endonuclease EcoRIat the 5′ end of the coding sequence and a BglII sequence at the 3′ end.Cleavage with BglII leaves a 5′ overhang or “sticky end” that iscompatible with the end generated by BamHI. The PCR-amplified DNA wasdigested with EcoRI and BamHI. The 2512 bp fragment containing thecoding region for the polymerase gene was gel purified and then ligatedinto a plasmid which contains an inducible promoter.

In one embodiment of the invention, the pTTQ18 vector, which containsthe hybrid trp-lac (tac) promoter, was used [M. J. R. Stark, Gene 5:255(1987)] and shown in FIG. 14. The tac promoter is under the control ofthe E. coli lac repressor. Repression allows the synthesis of the geneproduct to be suppressed until the desired level of bacterial growth hasbeen achieved, at which point repression is removed by addition of aspecific inducer, isopropyl-β-D-thiogalactopyranoside (IPTG). Such asystem allows the expression of foreign proteins that may slow orprevent growth of transformants.

Bacterial promoters, such as tac, may not be adequately suppressed whenthey are present on a multiple copy plasmid. If a highly toxic proteinis placed under control of such a promoter, the small amount ofexpression leaking through can be harmful to the bacteria. In anotherembodiment of the invention, another option for repressing synthesis ofa cloned gene product was used. The non-bacterial promoter, frombacteriophage T7, found in the plasmid vector series pET-3 was used toexpress the cloned mutant Taq polymerase genes [FIG. 15; Studier andMoffatt, J. Mol. Biol. 189:113 (1986)]. This promoter initiatestranscription only by T7 RNA polymerase. In a suitable strain, such asBL21(DE3)pLYS, the gene for this RNA polymerase is carried on thebacterial genome under control of the lac operator. This arrangement hasthe advantage that expression of the multiple copy gene (on the plasmid)is completely dependent on the expression of T7 RNA polymerase, which iseasily suppressed because it is present in a single copy.

For ligation into the pTTQ18 vector (FIG. 14), the PCR product DNAcontaining the Taq polymerase coding region (mutTaq, clone 4B, SEQ IDNO:21) was digested with EcoRI and BglII and this fragment was ligatedunder standard “sticky end” conditions [Sambrook et al. MolecularCloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp.1.63-1.69 (1989)] into the EcoRI and BamHI sites of the plasmid vectorpTTQ18. Expression of this construct yields a translational fusionproduct in which the first two residues of the native protein (Met-Arg)are replaced by three from the vector (Met-Asn-Ser), but the remainderof the natural protein would not change. The construct was transformedinto the JM109 strain of E. coli and the transformants were plated underincompletely repressing conditions that do not permit growth of bacteriaexpressing the native protein. These plating conditions allow theisolation of genes containing pre-existing mutations, such as those thatresult from the infidelity of Taq polymerase during the amplificationprocess.

Using this amplification/selection protocol, we isolated a clone(depicted in FIG. 4B) containing a mutated Taq polymerase gene (mutTaq,clone 4B). The mutant was first detected by its phenotype, in whichtemperature-stable 5′ nuclease activity in a crude cell extract wasnormal, but polymerization activity was almost absent (approximatelyless than 1% of wild type Taq polymerase activity).

DNA sequence analysis of the recombinant gene showed that it had changesin the polymerase domain resulting in two amino acid substitutions: an Ato G change at nucleotide position 1394 causes a Glu to Gly change atamino acid position 465 (numbered according to the natural nucleic andamino acid sequences, SEQ ID NOS:1 and 4) and another A to G change atnucleotide position 2260 causes a Gln to Arg change at amino acidposition 754. Because the Gln to Gly mutation is at a nonconservedposition and because the Glu to Arg mutation alters an amino acid thatis conserved in virtually all of the known Type A polymerases, thislatter mutation is most likely the one responsible for curtailing thesynthesis activity of this protein. The nucleotide sequence for the FIG.4B construct is given in SEQ ID NO:21. The enzyme encoded by thissequence is referred to as Cleavase® A/G.

Subsequent derivatives of DNAPTaq constructs were made from the mutTaqgene, thus, they all bear these amino acid substitutions in addition totheir other alterations, unless these particular regions were deleted.These mutated sites are indicated by black boxes at these locations inthe diagrams in FIG. 4. In FIG. 4, the designation “3′ Exo” is used toindicate the location of the 3′ exonuclease activity associated withType A polymerases which is not present in DNAPTaq. All constructsexcept the genes shown in FIGS. 4E, F and G were made in the pTTQ18vector.

The cloning vector used for the genes in FIGS. 4E and F was from thecommercially available pET-3 series, described above. Though this vectorseries has only a BamHI site for cloning downstream of the T7 promoter,the series contains variants that allow cloning into any of the threereading frames. For cloning of the PCR product described above, thevariant called pET-3c was used (FIG. 15). The vector was digested withBamHI, dephosphorylated with calf intestinal phosphatase, and the stickyends were filled in using the Klenow fragment of DNAPEc1 and dNTPs. Thegene for the mutant Taq DNAP shown in FIG. 4B (mutTaq, clone 4B) wasreleased from pTTQ18 by digestion with EcoRI and SalI, and the “stickyends” were filled in as was done with the vector. The fragment wasligated to the vector under standard blunt-end conditions (Sambrook etal., Molecular Cloning, supra), the construct was transformed into theBL21(DE3)pLYS strain of E. coli, and isolates were screened to identifythose that were ligated with the gene in the proper orientation relativeto the promoter. This construction yields another translational fusionproduct, in which the first two amino acids of DNAPTaq (Met-Arg) arereplaced by 13 from the vector plus two from the PCR primer(Met-Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg-Ile-Asn-Ser) (SEQ IDNO:29).

Our goal was to generate enzymes that lacked the ability to synthesizeDNA, but retained the ability to cleave nucleic acids with a 5′ nucleaseactivity. The act of primed, templated synthesis of DNA is actually acoordinated series of events, so it is possible to disable DNA synthesisby disrupting one event while not affecting the others. These stepsinclude, but are not limited to, primer recognition and binding, dNTPbinding and catalysis of the inter-nucleotide phosphodiester bond. Someof the amino acids in the polymerization domain of DNAPEcI have beenlinked to these functions, but the precise mechanisms are as yet poorlydefined.

One way of destroying the polymerizing ability of a DNA polymerase is todelete all or part of the gene segment that encodes that domain for theprotein, or to otherwise render the gene incapable of making a completepolymerization domain. Individual mutant enzymes may differ from eachother in stability and solubility both inside and outside cells. Forinstance, in contrast to the 5′ nuclease domain of DNAPEcI, which can bereleased in an active form from the polymerization domain by gentleproteolysis [Setlow and Kornberg, J. Biol. Chem. 247:232 (1972)], theThermus nuclease domain, when treated similarly, becomes less solubleand the cleavage activity is often lost.

Using the mutant gene shown in FIG. 4B as starting material, severaldeletion constructs were created. All cloning technologies were standard(Sambrook et al., supra) and are summarized briefly, as follows:

FIG. 4C: The mutTaq construct was digested with PstI, which cuts oncewithin the polymerase coding region, as indicated, and cuts immediatelydownstream of the gene in the multiple cloning site of the vector. Afterrelease of the fragment between these two sites, the vector wasre-ligated, creating an 894-nucleotide deletion, and bringing into framea stop codon 40 nucleotides downstream of the junction. The nucleotidesequence of this 5′ nuclease (clone 4C) is given in SEQ ID NO:9.

FIG. 4D: The mutTaq construct was digested with NheI, which cuts once inthe gene at position 2047. The resulting four-nucleotide 5′ overhangingends were filled in, as described above, and the blunt ends werere-ligated. The resulting four-nucleotide insertion changes the readingframe and causes termination of translation ten amino acids downstreamof the mutation. The nucleotide sequence of this 5′ nuclease (clone 4D)is given in SEQ ID NO: 10.

FIG. 4E: The entire mutTaq gene was cut from pTTQ18 using EcoRI and SalIand cloned into pET-3c, as described above. This clone was digested withBstXI and XcmI, at unique sites that are situated as shown in FIG. 4E.The DNA was treated with the Klenow fragment of DNAPEc1 and dNTPs, whichresulted in the 3′ overhangs of both sites being trimmed to blunt ends.These blunt ends were ligated together, resulting in an out-of-framedeletion of 1540 nucleotides. An in-frame termination codon occurs 18triplets past the junction site. The nucleotide sequence of this 5′nuclease (clone 4E) is given in SEQ ID NO:11, with the appropriateleader sequence given in SEQ ID NO:30. It is also referred to asCleavase® BX.

FIG. 4F: The entire mutTaq gene was cut from pTTQ18 using EcoRI and SalIand cloned into pET-3c, as described above. This clone was digested withBstXI and BamHI, at unique sites that are situated as shown in thediagram. The DNA was treated with the Klenow fragment of DNAPEc1 anddNTPs, which resulted in the 3′ overhang of the BstXI site being trimmedto a blunt end, while the 5′ overhang of the BamHI site was filled in tomake a blunt end. These ends were ligated together, resulting in anin-frame deletion of 903 nucleotides. The nucleotide sequence of the 5′nuclease (clone 4F) is given in SEQ ID NO: 12. It is also referred to asCleavas® BB.

FIG. 4G: This polymerase is a variant of that shown in FIG. 4E. It wascloned in the plasmid vector pET-21 (Novagen). The non-bacterialpromoter from bacteriophage T7, found in this vector, initiatestranscription only by T7 RNA polymerase. See Studier and Moffatt, supra.In a suitable strain, such as (DES)pLYS, the gene for this RNApolymerase is carried on the bacterial genome under control of the lacoperator. This arrangement has the advantage that expression of themultiple copy gene (on the plasmid) is completely dependent on theexpression of T7 RNA polymerase, which is easily suppressed because itis present in a single copy. Because the expression of these mutantgenes is under this tightly controlled promoter, potential problems oftoxicity of the expressed proteins to the host cells are less of aconcern.

The pET-21 vector also features a “His*Tag”, a stretch of sixconsecutive histidine residues that are added on the carboxy terminus ofthe expressed proteins. The resulting proteins can then be purified in asingle step by metal chelation chromatography, using a commericallyavailable (Novagen) column resin with immobilized Ni⁺⁺ ions. The 2.5 mlcolumns are reusable, and can bind up to 20 mg of the target proteinunder native or denaturing (guanidine*HCl or urea) conditions.

E. coli (DES)pLYS cells are transformed with the constructs describedabove using standard transformation techniques, and used to inoculate astandard growth medium (e.g., Luria-Bertani broth). Production of T7 RNApolymerase is induced during log phase growth by addition of IPTG andincubated for a further 12 to 17 hours. Aliquots of culture are removedboth before and after induction and the proteins are examined bySDS-PAGE. Staining with Coomassie Blue allows visualization of theforeign proteins if they account for about 3-5% of the cellular proteinand do not co-migrate with any of the major protein bands. Proteins thatco-migrate with major host protein must be expressed as more than 10% ofthe total protein to be seen at this tage of analysis.

Some mutant proteins are sequestered by the cells into inclusion bodies.These are granules that form in the cytoplasm when bacteria are made toexpress high levels of a foreign protein, and they can be purified froma crude lysate, and analyzed by SDS-PAGE to determine their proteincontent. If the cloned protein is found in the inclusion bodies, it mustbe released to assay the cleavage and polymerase activities. Differentmethods of solubilization may be appropriate for different proteins, anda variety of methods are known. See e.g., Builder & Ogez, U.S. Pat. No.4,511,502 (1985); Olson, U.S. Pat. No. 4,518,526 (1985); Olson & Pai,U.S. Pat. No. 4,511,503 (1985); Jones et al., U.S. Pat. No. 4,512,922(1985), all of which are hereby incorporated by reference.

The solubilized protein is then purified on the Ni⁺⁺ column as describedabove, following the manufacturers instructions (Novagen). The washedproteins are eluted from the column by a combination of imidazolecompetitor (1 M) and high salt (0.5 M NaCl), and dialyzed to exchangethe buffer and to allow denature proteins to refold. Typical recoveriesresult in approximately 20 μg of specific protein per ml of startingculture. The DNAP mutant is referred to as the Cleavase® BN nuclease andthe sequence is given in SEQ ID NO:31 (the amino acid sequence of theCleavase® BN nuclease is obtained by translating the DNA sequence of SEQID NO:31).

-   -   2. Modified DNAPTfl Gene

The DNA polymerase gene of Thermus flavus was isolated from the “T.flavus” AT-62 strain obtained from the American Type Tissue Collection(ATCC 33923). This strain has a different restriction map then does theT. flavus strain used to generate the sequence published by Akhmetzjanovand Vakhitov, supra. The published sequence is listed as SEQ ID NO:2. Nosequence data has been published for the DNA polymerase gene from theAT-62 strain of T. flavus.

Genomic DNA from T. flavus was amplified using the same primers used toamplify the T aquaticus DNA polymerase gene (SEQ ID NOS:13-14). Theapproximately 2500 base pair PCR fragment was digested with EcoRI andBamHI. The over-hanging ends were made blunt with the Klenow fragment ofDNAPEc1 and dNTPs. The resulting approximately 1800 base pair fragmentcontaining the coding region for the N-terminus was ligated into pET-3c,as described above. This construct, clone 5B, is depicted in FIG. 5B.The wild type T. flavus DNA polymerase gene is depicted in FIG. 5A. The5B clone has the same leader amino acids as do the DNAPTaq clones 4E andF which were cloned into pET-3c; it is not known precisely wheretranslation termination occurs, but the vector has a strongtranscription termination signal immediately downstream of the cloningsite.

B. Growth and Induction of Transformed Cells

Bacterial cells were transformed with the constructs described aboveusing standard transformation techniques and used to inoculate 2 mls ofa standard growth medium (e.g., Luria-Bertani broth). The resultingcultures were incubated as appropriate for the particular strain used,and induced if required for a particular expression system. For all ofthe constructs depicted in FIGS. 4 and 5, the cultures were grown to anoptical density (at 600nm wavelength) of 0.5 OD.

To induce expression of the cloned genes, the cultures were brought to afinal concentration of 0.4 mM IPTG and the incubations were continuedfor 12 to 17 hours. 50 μl aliquots of each culture were removed bothbefore and after induction and were combined with 20 μl of a standardgel loading buffer for sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). Subsequent staining with Coomassie Blue(Sambrook et al., supra) allows visualization of the foreign proteins ifthey account for about 3-5% of the cellular protein and do notco-migrate with any of the major E. coli protein bands. Proteins that doco-migrate with a major host protein must be expressed as more than 10%of the total protein to be seen at this stage of analysis.

C. Heat Lysis and Fractionation

Expressed thermostable proteins, i.e., the 5′ nucleases, were isolatedby heating crude bacterial cell extracts to cause denaturation andprecipitation of the less stable E. coli proteins. The precipitated E.coli proteins were then, along with other cell debris, removed bycentrifugation. 1.7 mls of the culture were pelleted bymicrocentrifugation at 12,000 to 14,000 rpm for 30 to 60 seconds. Afterremoval of the supernatant, the cells were resuspended in 400 μl ofbuffer A (50 mM Tris-HCl, pH 7.9, 50 mM dextrose, 1 mM EDTA),re-centrifuged, then resuspended in 80 μl of buffer A with 4mg/mllysozyme. The cells were incubated at room temperature for 15 minutes,then combined with 80 μl of buffer B (10 mnM Tris-HCl, pH 7.9, 50 mMKCl, 1 mM EDTA, 1 mM PMSF, 0.5% Tween-20, 0.5% Nonidet-P40).

This mixture was incubated at 75° C. for 1 hour to denature andprecipitate the host proteins. This cell extract was centrifuged at14,000 rpm for 15 minutes at 4° C., and the supernatant was transferredto a fresh tube. An aliquot of 0.5 to 1 μl of this supernatant was useddirectly in each test reaction, and the protein content of the extractwas determined by subjecting 7 μl to electrophoretic analysis, as above.The native recombinant Taq DNA polymerase [Englke, Anal. Biochem 191:396(1990)], and the double point mutation protein shown in FIG. 4B are bothsoluble and active at this point.

The foreign protein may not be detected after the heat treatments due tosequestration of the foreign protein by the cells into inclusion bodies.These are granules that form in the cytoplasm when bacteria are made toexpress high levels of a foreign protein, and they can be purified froma crude lysate, and analyzed SDS PAGE to determine their proteincontent. Many methods have been described in the literature, and oneapproach is described below.

D. Isolation and Solubilization of Inclusion Bodies

A small culture was grown and induced as described above. A 1.7 mlaliquot was pelleted by brief centrifugation, and the bacterial cellswere resuspended in 100 μl of Lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mMEDTA, 100 mM NaCl). 2.5 μl of 20 mM PMSF were added for a finalconcentration of 0.5 mM, and lysozyme was added to a concentration of1.0 mg/ml. The cells were incubated at room temperature for 20 minutes,deoxycholic acid was added to 1 mg/ml (1 μl of 100 mg/ml solution), andthe mixture was further incubated at 37° C. for about 15 minutes oruntil viscous. DNAse I was added to 10 μg/ml and the mixture wasincubated at room temperature for about 30 minutes or until it was nolonger viscous.

From this mixture the inclusion bodies were collected by centrifugationat 14,000 rpm for 15 minutes at 4° C., and the supernatant wasdiscarded. The pellet was resuspended in 100 μl of lysis buffer with 10mM EDTA (pH 8.0) and 0.5% Triton X-100. After 5 minutes at roomtemperature, the inclusion bodies were pelleted as before, and thesupernatant was saved for later analysis. The inclusion bodies wereresuspended in 50 μl of distilled water, and 5 μl was combined with SDSgel loading buffer (which dissolves the inclusion bodies) and analyzedelectrophoretically, along with an aliquot of the supernatant.

If the cloned protein is found in the inclusion bodies, it may bereleased to assay the cleavage and polymerase activities and the methodof solubilization must be compatible with the particular activity.Different methods of solubilization may be appropriate for differentproteins, and a variety of methods are discussed in Molecular Cloning(Sambrook et al., supra). The following is an adaptation we have usedfor several of our isolates.

20 μl of the inclusion body-water suspension were pelleted bycentrifugation at 14,000 rpm for 4 minutes at room temperature, and thesupernatant was discarded. To further wash the inclusion bodies, thepellet was resuspended in 20 μl of lysis buffer with 2M urea, andincubated at room temperature for one hour. The washed inclusion bodieswere then resuspended in 2 μl of lysis buffer with 8M urea; the solutionclarified visibly as the inclusion bodies dissolved. Undissolved debriswas removed by centrifugation at 14,000 rpm for 4 minutes at roomtemperature, and the extract supernatant was transferred to a freshtube.

To reduce the urea concentration, the extract was diluted into KH₂PO₄. Afresh tube was prepared containing 180 μl of 50 mM KH₂PO₄, pH 9.5, 1 mMEDTA and 50 mM NaCl. A 2 μl aliquot of the extract was added andvortexed briefly to mix. This step was repeated until all of the extracthad been added for a total of 10 additions. The mixture was allowed tosit at room temperature for 15 minutes, during which time someprecipitate often forms. Precipitates were removed by centrifugation at14,000 rpm, for 15 minutes at room temperature, and the supernatant wastransferred to a fresh tube. To the 200 μl of protein in the KH₂PO₄solution, 140-200 μl of saturated (NH₄)₂SO₄ were added, so that theresulting mixture was about 41% to 50% saturated (NH₄)₂SO₄. The mixturewas chilled on ice for 30 minutes to allow the protein to precipitate,and the protein was then collected by centrifugation at 14,000 rpm, for4 minutes at room temperature. The supernatant was discarded, and thepellet was dissolved in 20 μl Buffer C (20 mM HEPES, pH 7.9, 1 mM EDTA,0.5% PMSF, 25 mM KCl and 0.5% each of Tween-20 and Nonidet P 40). Theprotein solution was centrifuged again for 4 minutes to pellet insolublematerials, and the supernatant was removed to a fresh tube. The proteincontents of extracts prepared in this manner were visualized byresolving 1-4 μl by SDS-PAGE; 0.5 to 1 μl of extract was tested in thecleavage and polymerization assays as described.

E. Protein Analysis for Presence of Nuclease and Synthetic Activity

The 5′ nucleases described above and shown in FIGS. 4 and 5 wereanalyzed by the following methods.

-   -   1. Structure Specific Nuclease Assay

A candidate modified polymerase is tested for 5′ nuclease activity byexamining its ability to catalyze structure-specific cleavages. By theterm “cleavage structure” as used herein, is meant a nucleic acidstructure which is a substrate for cleavage by the 5′ nuclease activityof a DNAP.

The polymerase is exposed to test complexes that have the structuresshown in FIG. 16. Testing for 5′ nuclease activity involves threereactions: 1) a primer-directed cleavage (FIG. 16B) is performed becauseit is relatively insensitive to variations in the salt concentration ofthe reaction and can, therefore, be performed in whatever soluteconditions the modified enzyme requires for activity; this is generallythe same conditions preferred by unmodified polymerases; 2) a similarprimer-directed cleavage is performed in a buffer which permitsprimer-independent cleavage, i.e., a low salt buffer, to demonstratethat the enzyme is viable under these conditions; and 3) aprimer-independent cleavage (FIG. 16A) is performed in the same low saltbuffer.

The bifurcated duplex is formed between a substrate strand and atemplate strand as shown in FIG. 16. By the term “substrate strand” asused herein, is meant that strand of nucleic acid in which the cleavagemediated by the 5′ nuclease activity occurs. The substrate strand isalways depicted as the top strand in the bifurcated complex which servesas a substrate for 5′ nuclease cleavage (FIG. 16). By the term “templatestrand” as used herein, is meant the strand of nucleic acid which is atleast partially complementary to the substrate strand and which annealsto the substrate strand to form the cleavage structure. The templatestrand is always depicted as the bottom strand of the bifurcatedcleavage structure (FIG. 16). If a primer (a short oligonucleotide of 19to 30 nucleotides in length) is added to the complex, as whenprimer-dependent cleavage is to be tested, it is designed to anneal tothe 3′ arm of the template strand (FIG. 16B). Such a primer would beextended along the template strand if the polymerase used in thereaction has synthetic activity.

The cleavage structure may be made as a single hairpin molecule, withthe 3′ end of the target and the 5′ end of the pilot joined as a loop asshown in FIG. 16E. A primer oligonucleotide complementary to the 3′ armis also required for these tests so that the enzyme's sensitivity to thepresence of a primer may be tested.

Nucleic acids to be used to form test cleavage structures can bechemically synthesized, or can be generated by standard recombinant DNAtechniques. By the latter method, the hairpin portion of the moleculecan be created by inserting into a cloning vector duplicate copies of ashort DNA segment, adjacent to each other but in opposing orientation.The double-stranded fragment encompassing this inverted repeat, andincluding enough flanking sequence to give short (about 20 nucleotides)unpaired 5′ and 3′ arms, can then be released from the vector byrestriction enzyme digestion, or by PCR performed with an enzyme lackinga 5′ exonuclease (e.g., the Stoffel fragment of Amplitaq™ DNApolymerase, Vent™ DNA polymerase).

The test DNA can be labeled on either end, or internally, with either aradioisotope, or with a non-isotopic tag. Whether the hairpin DNA is asynthetic single strand or a cloned double strand, the DNA is heatedprior to use to melt all duplexes. When cooled on ice, the structuredepicted in FIG. 16E is formed, and is stable for sufficient time toperform these assays.

To test for primer-directed cleavage (Reaction 1), a detectable quantityof the test molecule (typically 1-100 fmol of ³²P-labeled hairpinmolecule) and a 10 to 100-fold molar excess of primer are placed in abuffer known to be compatible with the test enzyme. For Reaction 2,where primer-directed cleavage is performed under condition which allowprimer-independent cleavage, the same quantities of molecules are placedin a solution that is the same as the buffer used in Reaction 1regarding pH, enzyme stabilizers (e.g., bovine serum albumin, nonionicdetergents, gelatin) and reducing agents (e.g., dithiothreitol,2-mercaptoethanol) but that replaces any monovalent cation salt with 20mM KCl; 20 mM KCl is the demonstrated optimum for primer-independentcleavage. Buffers for enzymes, such as DNAPEc1, that usually operate inthe absence of salt are not supplemented to achieve this concentration.To test for primer-independent cleavage (Reaction 3) the same quantityof the test molecule, but no primer, are combined under the same bufferconditions used for Reaction 2.

All three test reactions are then exposed to enough of the enzyme thatthe molar ratio of enzyme to test complex is approximately 1:1. Thereactions are incubated at a range of temperatures up to, but notexceeding, the temperature allowed by either the enzyme stability or thecomplex stability, whichever is lower, up to 80° C. for enzymes fromthermophiles, for a time sufficient to allow cleavage (10 to 60minutes). The products of Reactions 1, 2 and 3 are resolved bydenaturing polyacrylamide gel electrophoresis, and visualized byautoradiography or by a comparable method appropriate to the labelingsystem used. Additional labeling systems include chemiluminescencedetection, silver or other stains, blotting and probing and the like.The presence of cleavage products is indicated by the presence ofmolecules which migrate at a lower molecular weight than does theuncleaved test structure. These cleavage products indicate that thecandidate polymerase has structure-specific 5′ nuclease activity.

To determine whether a modified DNA polymerase has substantially thesame 5′ nuclease activity as that of the native DNA polymerase, theresults of the above-described tests are compared with the resultsobtained from these tests performed with the native DNA polymerase. By“substantially the same 5′ nuclease activity” we mean that the modifiedpolymerase and the native polymerase will both cleave test molecules inthe same manner. It is not necessary that the modified polymerase cleaveat the same rate as the native DNA polymerase.

Some enzymes or enzyme preparations may have other associated orcontaminating activities that may be functional under the cleavageconditions described above and that may interfere with 5′ nucleasedetection. Reaction conditions can be modified in consideration of theseother activities, to avoid destruction of the substrate, or othermasking of the 5′ nuclease cleavage and its products. For example, theDNA polymerase I of E. coli (Pol I), in addition to its polymerase and5′ nuclease activities, has a 3′ exonuclease that can degrade DNA in a3′ to 5′ direction. Consequently, when the molecule in FIG. 16E isexposed to this polymerase under the conditions described above, the 3′exonuclease quickly removes the unpaired 3′ arm, destroying thebifurcated structure required of a substrate for the 5′ exonucleasecleavage and no cleavage is detected. The true ability of Pol I tocleave the structure can be revealed if the 3′ exonuclease is inhibitedby a change of conditions (e.g., pH), mutation, or by addition of acompetitor for the activity. Addition of 500 pmoles of a single-strandedcompetitor oligonucleotide, unrelated to the FIG. 16E structure, to thecleavage reaction with Pol I effectively inhibits the digestion of the3′ arm of the FIG. 16E structure without interfering with the 5′exonuclease release of the 5′ arm. The concentration of the competitoris not critical, but should be high enough to occupy the 3′ exonucleasefor the duration of the reaction.

Similar destruction of the test molecule may be caused by contaminantsin the candidate polymerase preparation. Several sets of the structurespecific nuclease reactions may be performed to determine the purity ofthe candidate nuclease and to find the window between under and overexposure of the test molecule to the polymerase preparation beinginvestigated.

The above described modified polymerases were tested for 5′ nucleaseactivity as follows: Reaction 1 was performed in a buffer of 10 mMTris-Cl, pH 8.5 at 20° C., 1.5 mM MgCl₂ and 50 mM KCl and in Reaction 2the KCl concentration was reduced to 20 mM. In Reactions 1 and 2, 10fmoles of the test substrate molecule shown in FIG. 16E were combinedwith 1 pmole of the indicated primer and 0.5 to 1.0 μl of extractcontaining the modified polymerase (prepared as described above). Thismixture was then incubated for 10 minutes at 55° C. For all of themutant polymerases tested these conditions were sufficient to givecomplete cleavage. When the molecule shown in FIG. 16E was labeled atthe 5′ end, the released 5′ fragment, 25 nucleotides long, wasconveniently resolved on a 20% polyacrylamide gel (19:1 cross-linked)with 7 M urea in a buffer containing 45 mM Tris-borate pH 8.3, 1.4 mMEDTA. Clones 4C-F and 5B exhibited structure-specific cleavagecomparable to that of the unmodified DNA polymerase. Additionally,clones 4E, 4F and 4G have the added ability to cleave DNA in the absenceof a 3′ arm as discussed above. Representative cleavage reactions areshown in FIG. 17.

For the reactions shown in FIG. 17, the mutant polymerase clones 4E (Taqmutant) and 5B (Tfl mutant) were examined for their ability to cleavethe hairpin substrate molecule shown in FIG. 16E. The substrate moleculewas labeled at the 5′ terminus with ³²P. 10 fmoles of heat-denatured,end-labeled substrate DNA and 0.5 units of DNAPTaq (lane 1) or 0.5 μl of4 e or 5 b extract (FIG. 17, lanes 2-7, extract was prepared asdescribed above) were mixed together in a buffer containing 10 mMTris-Cl, pH 8.5, 50 mM KCl and 1.5 MM MgCl₂. The final reaction volumewas 10 μl. Reactions shown in lanes 4 and 7 contain in addition 50 μM ofeach dNTP. Reactions shown in lanes 3, 4, 6 and 7 contain 0.2 μM of theprimer oligonucleotide (complementary to the 3′ arm of the substrate andshown in FIG. 16E). Reactions were incubated at 55° C. for 4 minutes.Reactions were stopped by the addition of 8 μl of 95% formamidecontaining 20 mM EDTA and 0.05% marker dyes per 10 μl reaction volume.Samples were then applied to 12% denaturing acrylamide gels. Followingelectrophoresis, the gels were autoradiographed. FIG. 17 shows thatclones 4E and 5B exhibit cleavage activity similar to that of the nativeDNAPTaq. Note that some cleavage occurs in these reactions in theabsence of the primer. When long hairpin structure, such as the one usedhere (FIG. 16E), are used in cleavage reactions performed in bufferscontaining 50 mM KCl a low level of primer-independent cleavage is seen.Higher concentrations of KCl suppress, but do not eliminate, thisprimer-independent cleavage under these conditions.

-   -   2. Assay F r Synthetic Activity

The ability of the modified enzyme or proteolytic fragments is assayedby adding the modified enzyme to an assay system in which a primer isannealed to a template and DNA synthesis is catalyzed by the addedenzyme. Many standard laboratory techniques employ such an assay. Forexample, nick translation and enzymatic sequencing involve extension ofa primer along a DNA template by a polymerase molecule.

In a preferred assay for determining the synthetic activity of amodified enzyme an oligonucleotide primer is annealed to asingle-stranded DNA template, e.g., bacteriophage M13 DNA, and theprimer/template duplex is incubated in the presence of the modifiedpolymerase in question, deoxynucleoside triphosphates (dNTPs) and thebuffer and salts known to be appropriate for the unmodified or nativeenzyme. Detection of either primer extension (by denaturing gelelectrophoresis) or dNTP incorporation (by acid precipitation orchromatography) is indicative of an active polymerase. A label, eitherisotopic or non-isotopic, is preferably included on either the primer oras a dNTP to facilitate detection of polymerization products. Syntheticactivity is quantified as the amount of free nucleotide incorporatedinto the growing DNA chain and is expressed as amount incorporated perunit of time under specific reaction conditions.

Representative results of an assay for synthetic activity is shown inFIG. 18. The synthetic activity of the mutant DNAPTaq clones 4B-F wastested as follows: A master mixture of the following buffer was made:1.2×PCR buffer (1×PCR buffer contains 50 mM KCl, 1.5 mM MgCl₂, 10 mMTris-Cl, ph 8.5 and 0.05% each Tween 20 and Nonidet P40), 50 μM each ofdGTP, DATP and dTTP, 5 μM dCTP and 0.125 μM α-³²P-dCTP at 600 Ci/mmol.Before adjusting this mixture to its final volume, it was divided intotwo equal aliquots. One received distilled water up to a volume of 50 μlto give the concentrations above. The other received 5 μg ofsingle-stranded M13mp18 DNA (approximately 2.5 pmol or 0.05 μM finalconcentration) and 250 pmol of M13 sequencing primer (5 μM finalconcentration) and distilled water to a final volume of 50 μl. Eachcocktail was warmed to 75° C. for 5 minutes and then cooled to roomtemperature. This allowed the primers to anneal to the DNA in theDNA-containing mixtures.

For each assay, 4 μl of the cocktail with the DNA was combined with 1 μlof the mutant polymerase, prepared as described, or 1 unit of DNAPTaq(Perkin Elmer) in 1 μl of dH₂O. A “no DNA” control was done in thepresence of the DNAPTaq (FIG. 18, lane 1), and a “no enzyme” control wasdone using water in place of the enzyme (lane 2). Each reaction wasmixed, then incubated at room temperature (approx. 22° C.) for 5minutes, then at 55° C. for 2 minutes, then at 72° C. for 2 minutes.This step incubation was done to detect polymerization in any mutantsthat might have optimal temperatures lower than 72° C. After the finalincubation, the tubes were spun briefly to collect any condensation andwere placed on ice. One μl of each reaction was spotted at an origin 1.5cm from the bottom edge of a polyethyleneimine (PEI) cellulose thinlayer chromatography plate and allowed to dry. The chromatography platewas run in 0.75 M NaH₂PO₄, pH 3.5, until the buffer front had runapproximately 9 cm from the origin. The plate was dried, wrapped inplastic wrap, marked with luminescent ink, and exposed to X-ray film.Incorporation was detected as counts that stuck where originallyspotted, while the unincorporated nucleotides were carried by the saltsolution from the origin.

Comparison of the locations of the counts with the two control lanesconfirmed the lack of polymerization activity in the mutantpreparations. Among the modified DNAPTaq clones, only clone 4B retainsany residual synthetic activity as shown in FIG. 18.

EXAMPLE 3 5′ Nucleases Derived from Thermostable DNA Polymerases CanCleave Short Hairpin Structures With Specificity

The ability of the 5′ nucleases to cleave hairpin structures to generatea cleaved hairpin structure suitable as a detection molecule wasexamined. The structure and sequence of the hairpin test molecule isshown in FIG. 19A (SEQ ID NO:15). The oligonucleotide (labeled “primer”in FIG. 19A, SEQ ID NO:22) is shown annealed to its complementarysequence on the 3′ arm of the hairpin test molecule. The hairpin testmolecule was single-end labeled with ³²P using a labeled T7 promoterprimer in a polymerase chain reaction. The label is present on the 5′arm of the hairpin test molecule and is represented by the star in FIG.19A.

The cleavage reaction was performed by adding 10 fmoles ofheat-denatured, end-labeled hairpin test molecule, 0.2 uM of the primeroligonucleotide (complementary to the 3′ arm of the hairpin), 50 μM ofeach dNTP and 0.5 units of DNAPTaq (Perkin Elmer) or 0.5 μl of extractcontaining a 5′ nuclease (prepared as described above) in a total volumeof 10 μl in a buffer containing 10 mM Tris-Cl, pH 8.5, 50 mM KCl and 1.5mM MgCl₂. Reactions shown in lanes 3, 5 and 7 were run in the absence ofdNTPs.

Reactions were incubated at 55° C. for 4 minutes. Reactions were stoppedat 55° C. by the addition of 8 μl of 95% formamide with 20 mM EDTA and0.05% marker dyes per 10 μl reaction volume. Samples were not heatedbefore loading onto denaturing polyacrylamide gels (10% polyacrylamide,19:1 crosslinking, 7 M urea, 89 mM Tris-borate, pH 8.3, 2.8 mM EDTA).The samples were not heated to allow for the resolution ofsingle-stranded and re-duplexed uncleaved hairpin molecules.

FIG. 19B shows that altered polymerases lacking any detectable syntheticactivity cleave a hairpin structure when an oligonucleotide is annealedto the single-stranded 3′ arm of the hairpin to yield a single speciesof cleaved product (FIG. 19B, lanes 3 and 4). 5′ nucleases, such asclone 4D, shown in lanes 3 and 4, produce a single cleaved product evenin the presence of dNTPs. 5′ nucleases which retain a residual amount ofsynthetic activity (less than 1% of wild type activity) produce multiplecleavage products as the polymerase can extend the oligonucleotideannealed to the 3′ arm of the hairpin thereby moving the site ofcleavage (clone 4B, lanes 5 and 6). Native DNATaq produces even morespecies of cleavage products than do mutant polymerases retainingresidual synthetic activity and additionally converts the hairpinstructure to a double-stranded form in the presence of dNTPs due to thehigh level of synthetic activity in the native polymerase (FIG. 19B,lane 8).

EXAMPLE 4 Test of the Trigger/Detection Assay

To test the ability of an oligonucleotide of the type released in thetrigger reaction of the trigger/detection assay to be detected in thedetection reaction of the assay, the two hairpin structures shown inFIG. 20A were synthesized using standard techniques. The two hairpinsare termed the A-hairpin (SEQ ID NO:23) and the T-hairpin (SEQ IDNO:24). The predicted sites of cleavage in the presence of theappropriate annealed primers are indicated by the arrows. The A- andT-hairpins were designed to prevent intra-strand mis-folding by omittingmost of the T residues in the A-hairpin and omitting most of the Aresidues in the T-hairpin. To avoid mis-priming and slippage, thehairpins were designed with local variations in the sequence motifs(e.g., spacing T residues one or two nucleotides apart or in pairs). TheA- and T-hairpins can be annealed together to form a duplex which hasappropriate ends for directional cloning in pUC-type vectors;restriction sites are located in the loop regions of the duplex and canbe used to elongate the stem regions if desired.

The sequence of the test trigger oligonucleotide is shown in FIG. 20B;this oligonucleotide is termed the alpha primer (SEQ ID NO:25). Thealpha primer is complementary to the 3′ arm of the T-hairpin as shown inFIG. 20A. When the alpha primer is annealed to the T-hairpin, a cleavagestructure is formed that is recognized by thermostable DNA polymerases.Cleavage of the T-hairpin liberates the 5′ single-stranded arm of theT-hairpin, generating the tau primer (SEQ ID NO:26) and a cleavedT-hairpin (FIG. 20B; SEQ ID NO:27). The tau primer is complementary tothe 3′ arm of the A-hairpin as shown in FIG. 20A. Annealing of the tauprimer to the A-hairpin generates another cleavage structure; cleavageof this second cleavage structure liberates the 5′ single-stranded armof the A-hairpin, generating another molecule of the alpha primer whichthen is annealed to another molecule of the T-hairpin. Thermocyclingreleases the primers so they can function in additional cleavagereactions. Multiple cycles of annealing and cleavage are carried out.The products of the cleavage reactions are primers and the shortenedhairpin structures shown in FIG. 20C. The shortened or cleaved hairpinstructures may be resolved from the uncleaved hairpins byelectrophoresis on denaturing acrylamide gels.

The annealing and cleavage reactions are carried as follows: In a 50 μlreaction volume containing 10 mM Tris-Cl, pH 8.5, 1.0 MgCl₂, 75 mM KCl,1 pmole of A-hairpin, 1 pmole T-hairpin, the alpha primer is added atequimolar amount relative to the hairpin structures (1 pmole) or atdilutions ranging from 10- to 10⁶-fold and 0.5 μl of extract containinga 5′ nuclease (prepared as described above) are added. The predictedmelting temperature for the alpha or trigger primer is 60° C. in theabove buffer. Annealing is performed just below this predicted meltingtemperature at 55° C. Using a Perkin Elmer DNA Thermal Cycler, thereactions are annealed at 55° C. for 30 seconds. The temperature is thenincreased slowly over a five minute period to 72° C. to allow forcleavage. After cleavage, the reactions are rapidly brought to 55° C.(1° C. per second) to allow another cycle of annealing to occur. A rangeof cycles are performed (20, 40 and 60 cycles) and the reaction productsare analyzed at each of these number of cycles. The number of cycleswhich indicates that the accumulation of cleaved hairpin products hasnot reached a plateau is then used for subsequent determinations when itis desirable to obtain a quantitative result.

Following the desired number of cycles, the reactions are stopped at 55°C. by the addition of 8 μl of 95% formamide with 20 mM EDTA and 0.05%marker dyes per 10 μl reaction volume. Samples are not heated beforeloading onto denaturing polyacrylamide gels (10% polyacrylamide, 19:1crosslinking, 7 M urea, 89 mM tris-borate, pH 8.3, 2.8 mM EDTA). Thesamples were not heated to allow for the resolution of single-strandedand re-duplexed uncleaved hairpin molecules.

The hairpin molecules may be attached to separate solid supportmolecules, such as agarose, styrene or magnetic beads, via the 3′ end ofeach hairpin. A spacer molecule may be placed between the 3′ end of thehairpin and the bead if so desired. The advantage of attaching thehairpins to a solid support is that this prevents the hybridization ofthe A- and T-hairpins to one another during the cycles of melting andannealing. The A- and T-hairpins are complementary to one another (asshown in FIG. 20D) and if allowed to anneal to one another over theirentire lengths this would reduce the amount of hairpins available forhybridization to the alpha and tau primers during the detectionreaction.

The 5′ nucleases of the present invention are used in this assay becausethey lack significant synthetic activity. The lack of synthetic activityresults in the production of a single cleaved hairpin product (as shownin FIG. 19B, lane 4). Multiple cleavage products may be generated by 1)the presence of interfering synthetic activity (see FIG. 19B, lanes 6and 8) or 2) the presence of primer-independent cleavage in thereaction. The presence of primer-independent cleavage is detected in thetrigger/detection assay by the presence of different sized products atthe fork of the cleavage structure. Primer-independent cleavage can bedampened or repressed, when present, by the use of uncleavablenucleotides in the fork region of the hairpin molecule. For example,thiolated nucleotides can be used to replace several nucleotides at thefork region to prevent primer-independent cleavage.

EXAMPLE 5 Cleavage of Linear Nucleic Acid Substrates

From the above, it should be clear that native (i.e., “wild type”)thermostable DNA polymerases are capable of cleaving hairpin structuresin a specific manner and that this discovery can be applied with successto a detection assay. In this example, the mutant DNAPs of the presentinvention are tested against three different cleavage structures shownin FIG. 22A. Structure 1 in FIG. 22A is simply single stranded 206-mer(the preparation and sequence information for which was discussedabove). Structures 2 and 3 are duplexes; structure 2 is the same hairpinstructure as shown in FIG. 12A (bottom), while structure 3 has thehairpin portion of structure 2 removed.

The cleavage reactions comprised 0.01 pmoles of the resulting substrateDNA, and 1 pmole of pilot oligonucleotide in a total volume of 10 μl of10 mM Tris-Cl, pH 8.3, 100 mM KCl, 1 mM MgCl₂. Reactions were incubatedfor 30 minutes at 55° C., and stopped by the addition of 8 μl of 95%formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to75° C. for 2 minutes immediately before electrophoresis through a 10%polyacrylamide gel (19:1 cross link), with 7M urea, in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA.

The results were visualized by autoradiography and are shown in FIG. 22Bwith the enzymes indicated as follows: I is native Taq DNAP; II isnative Tfl DNAP; III is Cleavase® BX shown in FIG. 4E; IV is Cleavase®BB shown in FIG. 4F; V is the mutant shown in FIG. 5B; and VI isCleavase® BN shown in FIG. 4G.

Structure 2 was used to “normalize” the comparison. For example, it wasfound that it took 50 ng of Taq DNAP and 300 ng of Cleavase® BN to givesimilar amounts of cleavage of Structure 2 in thirty (30) minutes. Underthese conditions native Taq DNAP is unable to cleave Structure 3 to anysignificant degree. Native Tfl DNAP cleaves Structure 3 in a manner thatcreates multiple products.

By contrast, all of the mutants tested cleave the linear duplex ofStructure 3. This finding indicates that this characteristic of themutant DNA polymerases is consistent of thermostable polymerases acrossthermophilic species.

The finding described herein that the mutant DNA polymerases of thepresent invention are capable of cleaving linear duplex structuresallows for application to a more straightforward assay design (FIG. 1A).FIG. 23 provides a more detailed schematic corresponding to the assaydesign of FIG. 1A.

The two 43-mers depicted in FIG. 23 were synthesized by standardmethods. Each included a fluorescein on the 5′ end for detectionpurposes and a biotin on the 3′ end to allow attachment to streptavidincoated paramagnetic particles (the biotin-avidin attachment is indicatedby “ ”).

Before the trityl groups were removed, the oligos were purified by HPLCto remove truncated by-products of the synthesis reaction. Aliquots ofeach 43-mer were bound to M-280 Dynabeads (Dynal) at a density of 100pmoles per mg of beads. Two (2) mgs of beads (200 μl) were washed twicein 1× wash/bind buffer (1 M NaCl, 5 mM Tris-Cl, pH 7.5, 0.5 mM EDTA)with 0.1% BSA, 200 μl per wash. The beads were magnetically sedimentedbetween washes to allow supernatant removal. After the second wash, thebeads were resuspended in 200 μl of 2× wash/bind buffer (2 M Na Cl, 10mM Tris-Cl, pH 7.5 with 1 mM EDTA), and divided into two 100 μlaliquots. Each aliquot received 1 μl of a 100 μM solution of one of thetwo oligonucleotides. After mixing, the beads were incubated at roomtemperature for 60 minutes with occasional gentle mixing. The beads werethen sedimented and analysis of the supernatants showed only traceamounts of unbound oligonucleotide, indicating successful binding. Eachaliquot of beads was washed three times, 100 μl per wash, with 1×wash/bind buffer, then twice in a buffer of 10 mM Tris-Cl, pH 8.3 and 75mM KCl. The beads were resuspended in a final volume of 100 μl of theTris/KCl, for a concentration of 1 pmole of oligo bound to 10 μg ofbeads per μl of suspension. The beads were stored at 4° C. between uses.

The types of beads correspond to FIG. 1A. That is to say, type 2 beadscontain the oligo (SEQ ID NO:33) comprising the complementary sequence(SEQ ID NO:34) for the alpha signal oligo (SEQ ID NO:35) as well as thebeta signal oligo (SEQ ID NO:36) which when liberated is a 24-mer. Thisoligo has no “As” and is “T” rich. Type 3 beads contain the oligo (SEQID NO:37) comprising the complementary sequence (SEQ ID NO:38) for thebeta signal oligo (SEQ ID NO:39) as well as the alpha signal oligo (SEQID NO:35) which when liberated is a 20-mer. This oligo has no “Ts” andis “A” rich.

Cleavage reactions comprised 1 μl of the indicated beads, 10 pmoles ofunlabelled alpha signal oligo as “pilot” (if indicated) and 500 ng ofCleavase® BN in 20 μl of 75 mM KCl, 10 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂and 10 μM CTAB. All components except the enzyme were assembled,overlaid with light mineral oil and warmed to 53° C. The reactions wereinitiated by the addition of prewarmed enzyme and incubated at thattemperature for 30 minutes. Reactions were stopped at temperature by theaddition of 16 μl of 95% formamide with 20 mM EDTA and 0.05% each ofbromophenol blue and xylene cyanol. This addition stops the enzymeactivity and, upon heating, disrupts the biotin-avidin link, releasingthe majority (greater than 95%) of the oligos from the beads. Sampleswere heated to 75° C. for 2 minutes immediately before electrophoresisthrough a 10% polyacrylamide gel (19:1 cross link), with 7 M urea, in abuffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. Results werevisualized by contact transfer of the resolved DNA to positively chargednylon membrane and probing of the blocked membrane with ananti-fluorescein antibody conjugated to alkaline phosphatase. Afterwashing, the signal was developed by incubating the membrane in WesternBlue (Promega) which deposits a purple precipitate where the antibody isbound.

FIG. 24 shows the propagation of cleavage of the linear duplex nucleicacid structures of FIG. 23 by the DNAP mutants of the present invention.The two center lanes contain both types of beads. As noted above, thebeta signal oligo (SEQ ID NO:36) when liberated is a 24-mer and thealpha signal oligo (SEQ ID NO:35) when liberated is a 20-mer. Theformation of the two lower bands corresponding to the 24-mer and 20-meris clearly dependent on “pilot”.

EXAMPLE 6 5′ Exonucleolytic Cleavage (“Nibbling”) by Thermostable DNAPs

It has been found that thermostable DNAPs, including those of thepresent invention, have a true 5′ exonuclease capable of nibbling the 5′end of a linear duplex nucleic acid structures. In this example, the 206base pair DNA duplex substrate is again employed (see above). In thiscase, it was produced by the use of one ³²P-labeled primer and oneunlabeled primer in a polymerase chain reaction. The cleavage reactionscomprised 0.01 pmoles of heat-denatured, end-labeled substrate DNA (withthe unlabeled strand also present), 5 pmoles of pilot oligonucleotide(see pilot oligos in FIG. 12A) and 0.5 units of DNAPTaq or 0.5μ ofCleavase® BB in the E. coli extract (see above), in a total volume of 10μl of 10 mM Tris •Cl, pH 8.5, 50 mM KCl, 1.5 mM MgCl₂.

Reactions were initiated at 65° C. by the addition of pre-warmed enzyme,then shifted to the final incubation temperature for 30 minutes. Theresults are shown in FIG. 25A. Samples in lanes 1-4 are the results withnative Taq DNAP, while lanes 5-8 shown the results with Cleavase® BB.The reactions for lanes 1, 2, 5, and 6 were performed at 65° C. andreactions for lanes 3, 4, 7, and 8 were performed at 50° C. and all werestopped at temperature by the addition of 8 μl of 95% formamide with 20mM EDTA and 0.05% marker dyes. Samples were heated to 75° C. for 2minutes immediately before electrophoresis through a 10% acrylamide gel(19:1 cross-linked), with 7 M urea, in a buffer of 45 mM Tris•Borate, pH8.3, 1.4 mM EDTA. The expected product in reactions 1, 2, 5, and 6 is 85nucleotides long; in reactions 3 and 7, the expected product is 27nucleotides long. Reactions 4 and 8 were performed without pilot, andshould remain at 206 nucleotides. The faint band seen at 24 nucleotidesis residual end-labeled primer from the PCR.

The surprising result is that Cleavase® BB under these conditions causesall of the label to appear in a very small species, suggesting thepossibility that the enzyme completely hydrolyzed the substrate. Todetermine the composition of the fastest-migrating band seen in lanes5-8 (reactions performed with the deletion mutant), samples of the 206base pair duplex were treated with either T7 gene 6 exonuclease (USB) orwith calf intestine alkaline phosphatase (Promega), according tomanufacturers' instructions, to produce either labeled mononucleotide(lane a of FIG. 25B) or free ³²P-labeled inorganic phosphate (lane b ofFIG. 25B), respectively. These products, along with the products seen inlane 7 of panel A were resolved by brief electrophoresis through a 20%acrylamide gel (19:1 cross-link), with 7 M urea, in a buffer of 45 mMTris•Borate, pH 8.3, 1.4 mM EDTA. Cleavase® BB is thus capable ofconverting the substrate to mononucleotides.

EXAMPLE 7 Nibbling is Duplex Dependent

The nibbling by Cleavase® BB is duplex dependent. In this example,internally labeled, single strands of the 206-mer were produced by 15cycles of primer extension incorporating α-³²P labeled dCTP combinedwith all four unlabeled dNTPs, using an unlabeled 206-bp fragment as atemplate. Single and double stranded products were resolved byelectrophoresis through a non-denaturing 6% polyacrylamide gel (29:1cross-link) in a buffer of 45 mM Tris•Borate, pH 8.3, 1.4 mM EDTA,visualized by autoradiography, excised from the gel, eluted by passivediffusion, and concentrated by ethanol precipitation.

The cleavage reactions comprised 0.04 pmoles of substrate DNA, and 2 μlof Cleavase® BB (in an E. coli extract as described above) in a totalvolume of 40 μl of 10 mM Tris•Cl, pH 8.5, 50 mM KCl, 1.5 mM MgCl₂.Reactions were initiated by the addition of pre-warmed enzyme; 10 μlaliquots were removed at 5, 10, 20, and 30 minutes, and transferred toprepared tubes containing 8 μl of 95% formamide with 30 mM EDTA and0.05% marker dyes. Samples were heated to 75° C. for 2 minutesimmediately before electrophoresis through a 10% acrylamide gel (19:1cross-linked), with 7 M urea, in a buffer of 45 mM Tris•Borate, pH 8.3,1.4 mM EDTA. Results were visualized by autoradiography as shown in FIG.26. Clearly, the cleavage by Cleavase® BB depends on a duplex structure;no cleavage of the single strand structure is detected whereas cleavageof the 206-mer duplex is complete.

EXAMPLE 8 Nibbling Can be Target Directed

The nibbling activity of the DNAPs of the present invention can beemployed with success in a detection assay. One embodiment of such anassay is shown in FIG. 27. In this assay, a labelled oligo is employedthat is specific for a target sequence. The oligo is in excess of thetarget so that hybridization is rapid. In this embodiment, the oligocontains two fluorescein labels whose proximity on the oligo causestheir emission to be quenched. When the DNAP is permitted to nibble theoligo the labels separate and are detectable. The shortened duplex isdestabilized and disassociates. Importantly, the target is now free toreact with an intact labelled oligo. The reaction can continue until thedesired level of detection is achieved. An analogous, althoughdifferent, type of cycling assay has been described employing lambdaexonuclease. See C. G. Copley and C. Boot, BioTechniques 13:888 (1992).

The success of such an assay depends on specificity. In other words, theoligo must hybridize to the specific target. It is also preferred thatthe assay be sensitive; the oligo ideally should be able to detect smallamounts of target. FIG. 28A shows a 5′-end ³²P-labelled primer bound toa plasmid target sequence. In this case, the plasmid was pUC19(commercially available) which was heat denatured by boiling two (2)minutes and then quick chilling. The primer is a 21-mer (SEQ ID NO:39).The enzyme employed was Cleavase® BX (a dilution equivalent to 5×10⁻³ μlextract) in 100 mM KCl, 10 mM Tris-Cl, pH 8.3, 2 mM MnCl₂. The reactionwas performed at 55° C. for sixteen (16) hours with or without genomicbackground DNA (from chicken blood). The reaction was stopped by theaddition of 8 μl of 95% formamide with 20 mM EDTA and marker dyes.

The products of the reaction were resolved by PAGE (10% polyacrylamide,19:1 cross link, 1×TBE) as seen in FIG. 28B. Lane “M” contains thelabelled 21-mer. Lanes 1-3 contain no specific target, although Lanes 2and 3 contain 100 ng and 200 ng of genomic DNA, respectively. Lanes 4, 5and 6 all contain specific target with either 0 ng, 100 ng or 200 ng ofgenomic DNA, respectively. It is clear that conversion tomononucleotides occurs in Lanes 4, 5 and 6 regardless of the presence oramount of background DNA. Thus, the nibbling can be target directed andspecific.

EXAMPLE 9 Cleavase Purification

As noted above, expressed thermostable proteins, ie., the 5′ nucleases,were isolated by crude bacterial cell extracts. The precipitated E. coliproteins were then, along with other cell debris, removed bycentrifugation. In this example, cells expressing the BN clone werecultured and collected (500 grams). For each gram (wet weight) of E.coli, 3 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 1 MM EDTA, 100 μMNaCl) was added. The cells were lysed with 200 μg/ml lysozyme at roomtemperature for 20 minutes. Thereafter deoxycholic acid was added tomake a 0.2% final concentration and the mixture was incubated 15 minutesat room temperature.

The lysate was sonicated for approximately 6-8 minutes at 0° C. Theprecipitate was removed by centrifugation (39,000 g for 20 minutes).Polyethyleneimine was added (0.5%) to the supernatant and the mixturewas incubated on ice for 15 minutes. The mixture was centrifuged (5,000g for 15 minutes) and the supernatant was retained. This was heated for30 minutes at 60° C. and then centrifuged again (5,000 g for 15 minutes)and the supernatant was again retained.

The supernatant was precipitated with 35% ammonium sulfate at 4° C. for15 minutes. The mixture was then centrifuged (5,000 g for 15 minutes)and the supernatant was removed. The precipitate was then dissolved in0.25 M KCl, 20 Tris pH 7.6, 0.2% Tween and 0.1 EDTA) and then dialyzedagainst Binding Buffer (8× Binding Buffer comprises: 40 mM imidazole, 4MNaCl, 160 mM Tris-HCl, pH 7.9).

The solubilized protein is then purified on the Ni⁺⁺ column (Novagen).The Binding Buffer is allows to drain to the top of the column bed andload the column with the prepared extract. A flow rate of about 10column volumes per hour is optimal for efficient purification. If theflow rate is too fast, more impurities will contaminate the elutedfraction.

The column is washed with 25 ml (10 volumes) of 1× Binding Buffer andthen washed with 15 ml (6 volumes) of 1× Wash Buffer (8× Wash Buffercomprises: 480 mM imidazole, 4M NaCl, 160 mM Tris-HCl, pH 7.9). Thebound protein was eluted with 15 ml (6 volumes) of 1× Elute Buffer (4×Elute Buffer comprises: 4 mM imidazole, 2 M NaCl, 80 mM Tris-HCl, pH7.9). Protein is then reprecipitated with 35% Ammonium Sulfate as above.The precipitate was then dissolved and dialyzed against: 20 mM Tris, 100mM KCl, 1 mM EDTA). The solution was brought up to 0.1% each of Tween 20and NP-40 and stored at 4° C.

EXAMPLE 10 The Use of Various Divalent Cations in the Cleavage ReactionInfluences the Nature of the Resulting Cleavage Products

In comparing the 5′ nucleases generated by the modification and/ordeletion of the C-terminal polymerization domain of Thermus aquaticusDNA polymerase (DNAPTaq), as diagrammed in FIG. 4B-G, significantdifferences in the strength of the interactions of these proteins withthe 3′ end of primers located upstream of the cleavage site (as depictedin FIG. 6) were noted. In describing the cleavage of these structures byPol I-type DNA polymerases [Example 1 and Lyamichev et al. (1993)Science 260:778], it was observed that in the absence of a primer, thelocation of the junction between the double-stranded region and thesingle-stranded 5′ and 3′ arms determined the site of cleavage, but inthe presence of a primer, the location of the 3′ end of the primerbecame the determining factor for the site of cleavage. It waspostulated that this affinity for the 3′ end was in accord with thesynthesizing function of the DNA polymerase.

Structure 2, shown in FIG. 22A, was used to test the effects of a 3′ endproximal to the cleavage site in cleavage reactions comprising severaldifferent solutions [e.g., solutions containing different salts (KCl orNaCl), different divalent cations (Mn²⁺ or Mg²⁺), etc.] as well as theuse of different temperatures for the cleavage reaction. When thereaction conditions were such that the binding of the enzyme (e.g., aDNAP comprising a 5′ nuclease, a modified DNAP or a 5′ nuclease) to the3′ end (of the pilot oligonucleotide) near the cleavage site was strong,the structure shown is cleaved at the site indicated in FIG. 22A. Thiscleavage releases the unpaired 5′ arm and leaves a nick between theremaining portion of the target nucleic acid and the folded 3′ end ofthe pilot oligonucleotide. In contrast, when the reaction conditions aresuch that the binding of the DNAP (comprising a 5′ nuclease) to the 3′end was weak, the initial cleavage was as described above, but after therelease of the 5′ arm, the remaining duplex is digested by theexonuclease function of the DNAP.

One way of weakening the binding of the DNAP to the 3′ end is to removeall or part of the domain to which at least some of this function hasbeen attributed. Some of 5′ nucleases created by deletion of thepolymerization domain of DNAPTaq have enhanced true exonucleasefunction, as demonstrated in Example 6.

The affinity of these types of enzymes (i.e., 5′ nucleases associatedwith or derived from DNAPs) for recessed 3′ ends may also be affected bythe identity of the divalent cation present in the cleavage reaction. Itwas demonstrated by Longley et al. [Nucl. Acids Res. 18:7317 (1990)]that the use of MnCl₂ in a reaction with DNAPTaq enabled the polymeraseto remove nucleotides from the 5′ end of a primer annealed to atemplate, albeit inefficiently. Similarly, by examination of thecleavage products generated using Structure 2 from FIG. 22A, asdescribed above, in a reaction containing either DNAPTaq or theCleavase® BB nuclease, it was observed that the substitution of MnCl₂for MgCl₂ in the cleavage reaction resulted in the exonucleolytic“nibbling” of the duplex downstream of the initial cleavage site. Whilenot limiting the invention to any particular mechanism, it is thoughtthat the substitution of MnCl₂ for MgCl₂ in the cleavage reactionlessens the affinity of these enzymes for recessed 3′ ends.

In all cases, the use of MnCl₂ enhances the 5′ nuclease function, and inthe case of the Cleavase® BB nuclease, a 50- to 100-fold stimulation ofthe 5′ nuclease function is seen. Thus, while the exonuclease activityof these enzymes was demonstrated above in the presence of MgCl₂, theassays described below show a comparable amount of exonuclease activityusing 50 to 100-fold less enzyme when MnCl₂ is used in place of MgCl₂.When these reduced amounts of enzyme are used in a reaction mixturecontaining MgCl₂, the nibbling or exonuclease activity is much lessapparent than that seen in Examples 6-8.

Similar effects are observed in the performance of the nucleic aciddetection assay described in Examples 11-18 below when reactionsperformed in the presence of either MgCl₂ or MnCl₂ are compared. In thepresence of either divalent cation, the presence of the invaderoligonucleotide (described below) forces the site of cleavage into theprobe duplex, but in the presence of MnCl₂ the probe duplex can befurther nibbled producing a ladder of products that are visible when a3′ end label is present on the probe oligonucleotide. When the invaderoligonucleotide is omitted from a reaction containing Mn²⁺, the probe isnibbled from the 5′ end. Mg² ⁺-based reactions display minimal nibblingof the probe oligonucleotide. In any of these cases, the digestion ofthe probe is dependent upon the presence of the target nucleic acid. Inthe examples below, the ladder produced by the enhanced nibblingactivity observed in the presence of Mn²⁺ is used as a positiveindicator that the probe oligonucleotide has hybridized to the targetsequence.

EXAMPLE 11 Invasive 5′ Endonucleolytic Cleavage by Thermostable 5′Nucleases in the Absence of Polymerization

As described in the examples above, 5′ nucleases cleave near thejunction between single-stranded and base-paired regions in a bifurcatedduplex, usually about one base pair into the base-paired region. In thisexample, it is shown that thermostable 5′ nucleases, including those ofthe present invention (e.g., Cleavase® BN nuclease, Cleavase® A/Gnuclease), have the ability to cleave a greater distance into the basepaired region when provided with an upstream oligonucleotide bearing a3′ region that is homologous to a 5′ region of the subject duplex, asshown in FIG. 30.

FIG. 30 shows a synthetic oligonucleotide which was designed to foldupon itself which consists of the following sequence:5′-GTTCTCTGCTCTCTGGTCGCTG TCTCGCTTGTGAAACAAGCGAGACAGCGTGGTCTCTCG-3′ (SEQID NO:40). This oligonucleotide is referred to as the “S-60 Hairpin.”The 15 basepair hairpin formed by this oligonucleotide is furtherstabilized by a “tri-loop” sequence in the loop end (i.e., threenucleotides form the loop portion of the hairpin) [Hiraro, I. et al.(1994) Nucleic Acids Res. 22(4):576]. FIG. 30 also show the sequence ofthe P-15 oligonucleotide and the location of the region ofcomplementarity shared by the P-15 and S-60 hairpin oligonucleotides.The sequence of the P-15 oligonucleotide is 5′-CGAGAGACCACGCTG-3′ (SEQID NO:41). As discussed in detail below, the solid black arrowheadsshown in FIG. 29 indicate the sites of cleavage of the S-60 hairpin inthe absence of the P-15 oligonucleotide and the hollow arrow headsindicate the sites of cleavage in the presence of the P-15oligonucleotide. The size of the arrow head indicates the relativeutilization of a particular site.

The S-60 hairpin molecule was labeled on its 5′ end with biotin forsubsequent detection. The S-60 hairpin was incubated in the presence ofa thermostable 5′ nuclease in the presence or the absence of the P-15oligonucleotide. The presence of the full duplex which can be formed bythe S-60 hairpin is demonstrated by cleavage with the Cleavase® BN 5′nuclease, in a primer-independent fashion (i.e., in the absence of theP-15 oligonucleotide). The release of 18 and 19-nucleotide fragmentsfrom the 5′ end of the S-60 hairpin molecule showed that the cleavageoccurred near the junction between the single and double strandedregions when nothing is hybridized to the 3′ arm of the S-60 hairpin(FIG. 31, lane 2).

The reactions shown in FIG. 31 were conducted as follows. Twenty fmoleof the 5′ biotin-labeled hairpin DNA (SEQ ID NO:40) was combined with0.1 ng of Cleavase® BN enzyme and 1 μl of 100 mM MOPS (pH 7.5)containing 0.5% each of Tween-20 and NP-40 in a total volume of 9 μl. Inthe reaction shown in lane 1, the enzyme was omitted and the volume wasmade up by addition of distilled water (this served as the uncut or noenzyme control). The reaction shown in lane 3 of FIG. 31 also included0.5 pmole of the P15 oligonucleotide (SEQ ID NO:41), which can hybridizeto the unpaired 3′ arm of the S-60 hairpin (SEQ ID NO:40), as diagrammedin FIG. 30.

The reactions were overlaid with a drop of mineral oil, heated to 95° C.for 15 seconds, then cooled to 37° C., and the reaction was started bythe addition of 1 μl of 10 mM MnCl₂ to each tube. After 5 minutes, thereactions were stopped by the addition of 6 μl of 95% formamidecontaining 20 mM EDTA and 0.05% marker dyes. Samples were heated to 75°C. for 2 minutes immediately before electrophoresis through a 15%acrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated allowing the gel toremain flat on one plate. A 0.2 mm-pore positively-charged nylonmembrane (NYTRAN, Schleicher and Schuell, Keene, N.H.), pre-wetted inH₂O, was laid on top of the exposed gel. All air bubbles were removed.Two pieces of 3MM filter paper (Whatman) were then placed on top of themembrane, the other glass plate was replaced, and the sandwich wasclamped with binder clips. Transfer was allowed to proceed overnight.After transfer, the membrane was carefully peeled from the gel andallowed to air dry. After complete drying, the membrane was washed in1.2× Sequenase Images Blocking Buffer (United States Biochemical) using0.3 ml of buffer/cm² of membrane. The wash was performed for 30 minutesat room temperature. A streptavidin-alkaline phosphatase conjugate(SAAP, United States Biochemical) was added to a 1:4000 dilutiondirectly to the blocking solution, and agitated for 15 minutes. Themembrane was rinsed briefly with H₂O and then washed three times for 5minutes per wash using 0.5 ml/cm² of 1×SAAP buffer (100 mM Tris-HCl, pH10, 50 mM NaCl) with 0.1% sodium dodecyl sulfate (SDS). The membrane wasrinsed briefly with H₂O between each wash. The membrane was then washedonce in 1×SAAP buffer containing 1 mM MgCl₂ without SDS, drainedthoroughly and placed in a plastic heat-sealable bag. Using a sterilepipet, 5 mls of CDP-Star™ (Tropix, Bedford, Mass.) chemiluminescentsubstrate for alkaline phosphatase were added to the bag and distributedover the entire membrane for 2-3 minutes. The CDP-Star™ -treatedmembrane was exposed to XRP X-ray film (Kodak) for an initial exposureof 10 minutes.

The resulting autoradiograph is shown in FIG. 31. In FIG. 31, the lanelabelled “M” contains the biotinylated P-15 oligonucleotide which servedas a marker. The sizes (in nucleotides) of the uncleaved S-60 hairpin(60 nuc; lane 1), the marker (15 nuc; lane “M”) and the cleavageproducts generated by cleavage of the S-60 hairpin in the presence (lane3) or absence (lane 2) of the P-15 oligonucleotide are indicated.

Because the complementary regions of the S-60 hairpin are located on thesame molecule, essentially no lag time should be needed to allowhybridization (i.e., to form the duplex region of the hairpin). Thishairpin structure would be expected to form long before the enzyme couldlocate and cleave the molecule. As expected, cleavage in the absence ofthe primer oligonucleotide was at or near the junction between theduplex and single-stranded regions, releasing the unpaired 5′ arm (FIG.31, lane 2). The resulting cleavage products were 18 and 19 nucleotidesin length.

It was expected that stability of the S-60 hairpin with the tri-loopwould prevent the P-15 oligonucleotide from promoting cleavage in the“primer-directed” manner described in Example 1 above, because the 3′end of the “primer” would remain unpaired. Surprisingly, it was foundthat the enzyme seemed to mediate an “invasion” by the P-15 primer intothe duplex region of the S-60 hairpin, as evidenced by the shifting ofthe cleavage site 3 to 4 basepairs further into the duplex region,releasing the larger products (22 and 21 nuc.) observed in lane 3 ofFIG. 31.

The precise sites of cleavage of the S-60 hairpin are diagrammed on thestructure in FIG. 30, with the solid black arrowheads indicating thesites of cleavage in the absence of the P-15 oligonucleotide and thehollow arrow heads indicating the sites of cleavage in the presence ofP-15.

These data show that the presence on the 3′ arm of an oligonucleotidehaving some sequence homology with the first several bases of thesimilarly oriented strand of the downstream duplex can be a dominantfactor in determining the site of cleavage by 5′ nucleases. Because theoligonucleotide which shares some sequence homology with the firstseveral bases of the similarly oriented strand of the downstream duplexappears to invade the duplex region of the hairpin, it is referred to asan “invader” oligonucleotide. As shown in the examples below, an invaderoligonucleotide appears to invade (or displace) a region of duplexednucleic acid regardless of whether the duplex region is present on thesame molecule (i.e., a hairpin) or whether the duplex is formed betweentwo separate nucleic acid strands.

EXAMPLE 12 The Invader Oligonucleotide Shifts the Site of Cleavage in aPre-Formed Probe/Target Duplex

In Example 11 it was demonstrated that an invader oligonucleotide couldshift the site at which a 5′ nuclease cleaves a duplex region present ona hairpin molecule. In this example, the ability of an invaderoligonucleotide to shift the site of cleavage within a duplex regionformed between two separate strands of nucleic acid molecules wasexamined.

A single-stranded target DNA comprising the single-stranded circularM13mp19 molecule and a labeled (fluorescein) probe oligonucleotide weremixed in the presence of the reaction buffer containing salt (KCl) anddivalent cations (Mg²⁺ or Mn²⁺) to promote duplex formation. The probeoligonucleotide refers to a labelled oligonucleotide which iscomplementary to a region along the target molecule (e.g., M13mp19). Asecond oligonucleotide (unlabelled) was added to the reaction after theprobe and target had been allowed to anneal. The second oligonucleotidebinds to a region of the target which is located downstream of theregion to which the probe oligonucleotide binds. This secondoligonucleotide contains sequences which are complementary to a secondregion of the target molecule. If the second oligonucleotide contains aregion which is complementary to a portion of the sequences along thetarget to which the probe oligonucleotide also binds, this secondoligonucleotide is referred to as an invader oligonucleotide (see FIG.32 c).

FIG. 32 depicts the annealing of two oligonucleotides to regions alongthe M13mp19 target molecule (bottom strand in all three structuresshown). In FIG. 32 only a 52 nucleotide portion of the M13mp19 moleculeis shown; this 52 nucleotide sequence is listed in SEQ ID NO:42. Theprobe oligonucleotide contains a fluorescein label at the 3′ end; thesequence of the probe is 5′-AGAAAGGAAGGGAAGAAAGC GAAAGG-3′ (SEQ IDNO:43). In FIG. 32, sequences comprising the second oligonucleotide,including the invader oligonucleotide are underlined. In FIG. 32 a, thesecond oligonucleotide, which has the sequence 5′-GACGGGGAAAGCCGGCGAACG-3′ (SEQ ID NO:44), is complementary to a different and downstreamregion of the target molecule than is the probe oligonucleotide (labeledwith fluorescein or “Fluor”); there is a gap between the second,upstream oligonucleotide and the probe for the structure shown in FIG.32 a. In FIG. 32 b, the second, upstream oligonucleotide, which has thesequence 5′-GAAAGCCGGCGAACGTGGCG-3′ (SEQ ID NO:45), is complementary toa different region of the target molecule than is the probeoligonucleotide, but in this case, the second oligonucleotide and theprobe oligonucleotide abut one another (that is the 3′ end of thesecond, upstream oligonucleotide is immediately adjacent to the 5′ endof the probe such that no gap exists between these twooligonucleotides). In FIG. 32 c, the second, upstream oligonucleotide[5′-GGCGAACGTGGCGAGAAAGGA-3′ (SEQ ID NO:46)] and the probeoligonucleotide share a region of complementarity with the targetmolecule. Thus, the upstream oligonucleotide has a 3′ arm which has asequence identical to the first several bases of the downstream probe.In this situation, the upstream oligonucleotide is referred to as an“invader” oligonucleotide.

The effect of the presence of an invader oligonucleotide upon thepattern of cleavage in a probe/target duplex formed prior to theaddition of the invader was examined. The invader oligonucleotide andthe enzyme were added after the probe was allowed to anneal to thetarget and the position and extent of cleavage of the probe wereexamined to determine a) if the invader was able to shift the cleavagesite to a specific internal region of the probe, and b), if the reactioncould accumulate specific cleavage products over time, even in theabsence of thermal cycling, polymerization, or exonuclease removal ofthe probe sequence.

The reactions were carried out as follows. Twenty μl each of two enzymemixtures were prepared, containing 2 μl of Cleavase® A/G nucleaseextract (prepared as described in Example 2), with or without 50 pmoleof the invader oligonucleotide (SEQ ID NO:46), as indicated, per 4 μl ofthe mixture. For each of the eight reactions shown in FIG. 33, 150 fmoleof M13mp19 single-stranded DNA (available from Life Technologies, Inc.)was combined with 5 pmoles of fluorescein labeled probe (SEQ ID NO:43),to create the structure shown in FIG. 31 c, but without the invaderoligonucleotide present (the probe/target mixture). One half (4 tubes)of the probe/target mixtures were combined with 1 μl of 100 mM MOPS, pH7.5 with 0.5% each of Tween-20 and NP-40, 0.5 μl of 1 M KCl and 0.25 μlof 80 mM MnCl₂, and distilled water to a volume of 6 μl. The second setof probe/target mixtures were combined with 1 μl of 100 mM MOPS, pH 7.5with 0.5% each of Tween-20 and NP-40, 0.5 μl of 1 M KCl and 0.25 μl of80 mM MgCl₂. The second set of mixtures therefore contained MgCl₂ inplace of the MnCl₂ present in the first set of mixtures.

The mixtures (containing the probe/target with buffer, KCl and divalentcation) were covered with a drop of ChillOut® evaporation barrier (MJResearch) and were brought to 60° C. for 5 minutes to allow annealing.Four μl of the above enzyme mixtures without the invader oligonucleotidewas added to reactions whose products are shown in lanes 1, 3, 5 and 7of FIG. 33. Reactions whose products are shown lanes 2, 4, 6, and 8 ofFIG. 33 received the same amount of enzyme mixed with the invaderoligonucleotide (SEQ ID NO:46). Reactions 1, 2, 5 and 6 were incubatedfor 5 minutes at 60° C. and reactions 3, 4, 7 and 8 were incubated for15 minutes at 60° C.

All reactions were stopped by the addition of 8 μl of 95% formamide with20 mM EDTA and 0.05% marker dyes. Samples were heated to 90° C. for 1minute immediately before electrophoresis through a 20% acrylamide gel(19:1 cross-linked), containing 7 M urea, in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA. Following electrophoresis, thereaction products and were visualized by the use of an Hitachi FMBIOfluorescence imager, the output of which is seen in FIG. 33. The verylow molecular weight fluorescent material seen in all lanes at or nearthe salt front in FIG. 33 and other fluoro-imager figures is observedwhen fluorescently-labeled oligonucleotides are electrophoresed andimaged on a fluoro-imager. This material is not a product of thecleavage reaction.

The use of MnCl₂ in these reactions (lanes 1-4) stimulates the trueexonuclease or “nibbling” activity of the Cleavase® enzyme, as describedin Example 7, as is clearly seen in lanes 1 and 3 of FIG. 33. Thisnibbling of the probe oligonucleotide (SEQ ID NO:43) in the absence ofinvader oligonucleotide (SEQ ID NO:46) confirms that the probeoligonucleotide is forming a duplex with the target sequence. Theladder-like products produced by this nibbling reaction may be difficultto differentiate from degradation of the probe by nucleases that mightbe present in a clinical specimen. In contrast, introduction of theinvader oligonucleotide (SEQ ID NO:46) caused a distinctive shift in thecleavage of the probe, pushing the site of cleavage 6 to 7 bases intothe probe, confirming the annealing of both oligonucleotides. Inpresence of MnCl₂, the exonuclease “nibbling” may occur after theinvader-directed cleavage event, until the residual duplex isdestabilized and falls apart.

In a magnesium based cleavage reaction (lanes 5-8), the nibbling or trueexonuclease function of the Cleavase® A/G is enzyme suppressed (but theendonucleolytic function of the enzyme is essentially unaltered), so theprobe oligonucleotide is not degraded in the absence of the invader(FIG. 33, lanes 5 and 7). When the invader is added, it is clear thatthe invader oligonucleotide can promote a shift in the site of theendonucleolytic cleavage of the annealed probe. Comparison of theproducts of the 5 and 15 minute reactions with invader (lanes 6 and 8 inFIG. 33) shows that additional probe hybridizes to the target and iscleaved. The calculated melting temperature (T_(m)) of the portion ofprobe that is not invaded (i.e., nucleotides 9-26 of SEQ ID NO:43) is56° C., so the observed turnover (as evidenced by the accumulation ofcleavage products with increasing reaction time) suggests that the fulllength of the probe molecule, with a calculated T_(m) of 76° C., is mustbe involved in the subsequent probe annealing events in this 60° C.reaction.

EXAMPLE 13 The Overlap of the 3′ Invader Oligonucleotide Sequence withthe 5′ Region of the Probe Causes a Shift in the Site of Cleavage

In Example 12, the ability of an invader oligonucleotide to cause ashift in the site of cleavage of a probe annealed to a target moleculewas demonstrated. In this example, experiments were conducted to examinewhether the presence of an oligonucleotide upstream from the probe wassufficient to cause a shift in the cleavage site(s) along the probe orwhether the presence of nucleotides on the 3′ end of the invaderoligonucleotide which have the same sequence as the first severalnucleotides at the 5′ end of the probe oligonucleotide were required topromote the shift in cleavage.

To examine this point, the products of cleavage obtained from threedifferent arrangements of target-specific oligonucleotides are compared.A diagram of these oligonucleotides and the way in which they hybridizeto a test nucleic acid, M13mp19, is shown in FIG. 32. In FIG. 32 a, the3′ end of the upstream oligonucleotide (SEQ ID NO:45) is locatedupstream of the 5′ end of the downstream “probe” oligonucleotide (SEQ IDNO:43) such that a region of the M13 target which is not paired toeither oligonucleotide is present. In FIG. 32 b, the sequence of theupstream oligonucleotide (SEQ ID NO:45) is immediately upstream of theprobe (SEQ ID NO:43), having neither a gap nor an overlap between thesequences. FIG. 32 c diagrams the arrangement of the substrates used inthe assay of the present invention, showing that the upstream “invader”oligonucleotide (SEQ ID NO:46) has the same sequence on a portion of its3′ region as that present in the 5′ region of the downstream probe (SEQID NO:43). That is to say, these regions will compete to hybridize tothe same segment of the M13 target nucleic acid.

In these experiments, four enzyme mixtures were prepared as follows(planning 5 μl per digest): Mixture 1 contained 2.25 μl of Cleavase® A/Gnuclease extract (prepared as described in Example 2) per 5 μl ofmixture, in 20 mM MOPS, pH 7.5 with 0.1% each of Tween 20 and NP-40, 4mM MnCl₂ and 100 mM KCl. Mixture 2 contained 11.25 units of Taq DNApolymerase (Promega Corp., Madison, Wis.) per 5 μl of mixture in 20 mMMOPS, pH 7.5 with 0.1% each of Tween 20 and NP-40, 4 mM MnCl₂ and 100 mMKCl. Mixture 3 contained 2.25 μl of Cleavase® A/G nuclease extract per 5μl of mixture in 20 mM Tris-HCl, pH 8.5, 4 mM MgCl₂ and 100 mM KCl.Mixture 4 contained 11.25 units of Taq DNA polymerase per 5 μl ofmixture in 20 mM Tris-HCl, pH 8.5, 4 mM MgCl₂ and 100 mM KCl.

For each reaction, 50 fmole of M13mp19 single-stranded DNA (the targetnucleic acid) was combined with 5 pmole of the probe oligonucleotide(SEQ ID NO:43 which contained a fluorescein label at the 3′ end) and 50pmole of one of the three upstream oligonucleotides diagrammed in FIG.32 (i.e., one of SEQ ID NOS:44-46), in a total volume of 5 μl ofdistilled water. The reactions were overlaid with a drop of ChillOut™evaporation barrier (MJ Research) and warmed to 62° C. The cleavagereactions were started by the addition of 5 μl of an enzyme mixture toeach tube, and the reactions were incubated at 62° C. for 30 min. Thereactions shown in lanes 1-3 of FIG. 34 received Mixture 1; reactions4-6 received Mixture 2; reactions 7-9 received Mixture 3 and reactions10-12 received Mixture 4.

After 30 minutes at 62° C., the reactions were stopped by the additionof 8 μl of 95% formamide with 20 mM EDTA and 0.05% marker dyes. Sampleswere heated to 75° C. for 2 minutes immediately before electrophoresisthrough a 20% acrylamide gel (19:1 cross-linked), with 7 M urea, in abuffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

Following electrophoresis, the products of the reactions were visualizedby the use of an Hitachi FMBIO fluorescence imager, the output of whichis seen in FIG. 34. The reaction products shown in lanes 1, 4, 7 and 10of FIG. 34 were from reactions which contained SEQ ID NO:44 as theupstream oligonucleotide (see FIG. 32 a). The reaction products shown inlanes 2, 5, 8 and 11 of FIG. 34 were from reactions which contained SEQID NO:45 as the upstream oligonucleotide (see FIG. 32 b). The reactionproducts shown in lanes 3, 6, 9 and 12 of FIG. 34 were from reactionswhich contained SEQ ID NO:46, the invader oligonucleotide, as theupstream oligonucleotide (see FIG. 32 c).

Examination of the Mn²⁺ based reactions using either Cleavase® A/Gnuclease or DNAPTaq as the cleavage agent (lanes 1 through 3 and 4through 6, respectively) shows that both enzymes have active exonucleasefunction in these buffer conditions. The use of a 3′ label on the probeoligonucleotide allows the products of the nibbling activity to remainlabeled, and therefore visible in this assay. The ladders seen in lanes1, 2, 4 and 5 confirm that the probe hybridize to the target DNA asintended. These lanes also show that the location of the non-invasiveoligonucleotides have little effect on the products generated. Theuniform ladder created by these digests would be difficult todistinguish from a ladder causes by a contaminating nuclease, as onemight find in a clinical specimen. In contrast, the products displayedin lanes 3 and 6, where an invader oligonucleotide was provided todirect the cleavage, show a very distinctive shift, so that the primarycleavage product is smaller than those seen in the non-invasivecleavage. This product is then subject to further nibbling in theseconditions, as indicated by the shorter products in these lanes. Theseinvader-directed cleavage products would be easily distinguished from abackground of non-specific degradation of the probe oligonucleotide.

When Mg⁺ is used as the divalent cation the results are even moredistinctive. In lanes 7, 8, 10 and 11 of FIG. 34, where the upstreamoligonucleotides were not invasive, minimal nibbling is observed. Theproducts in the DNAPTaq reactions show some accumulation of probe thathas been shortened on the 5′ end by one or two nucleotides consistentwith previous examination of the action of this enzyme on nickedsubstrates (Longley et al., supra). When the upstream oligonucleotide isinvasive, however, the appearance of the distinctively shifted probeband is seen. These data clearly indicated that it is the invasive 3′portion of the upstream oligonucleotide that is responsible for fixingthe site of cleavage of the downstream probe.

Thus, the above results demonstrate that it is the presence of the freeor initially non-annealed nucleotides at the 3′ end of the invaderoligonucleotide which mediate the shift in the cleavage site, not justthe presence of an oligonucleotide annealed upstream of the probe.Nucleic acid detection assays which employ the use of an invaderoligonucleotide are termed “invader-directed cleavage” assays.

EXAMPLE 14 Invader-Directed Cleavage Recognizes Single and DoubleStranded Target Molecules in a Background of Non-Target DNA Molecules

For a nucleic acid detection method to be broadly useful, it must beable to detect a specific target in a sample that may contain largeamounts of other DNA, e.g., bacterial or human chromosomal DNA. Theability of the invader directed cleavage assay to recognize and cleaveeither single- or double-stranded target molecules in the presence oflarge amounts of non-target DNA was examined. In these experiments amodel target nucleic acid, M13, in either single or double stranded form(single-stranded M13mp18 is available from Life Technologies, Inc anddouble-stranded M13mp19 is available from New England Biolabs), wascombined with human genomic DNA (Novagen, Madison, Wis.) and thenutilized in invader-directed cleavage reactions. Before the start of thecleavage reaction, the DNAs were heated to 95° C. for 15 minutes tocompletely denature the samples, as is standard practice in assays, suchas polymerase chain reaction or enzymatic DNA sequencing, which involvesolution hybridization of oligonucleotides to double-stranded targetmolecules.

For each of the reactions shown in lanes 2-5 of FIG. 35, the target DNA(25 fmole of the ss DNA or 1 pmole of the ds DNA) was combined with 50pmole of the invader oligonucleotide (SEQ ID NO:46); for the reactionshown in lane 1 the target DNA was omitted. Reactions 1, 3 and 5 alsocontained 470 ng of human genomic DNA. These mixtures were brought to avolume of 10 μl with distilled water, overlaid with a drop of ChillOut™evaporation barrier (MJ Research), and brought to 95° C. for 15 minutes.After this incubation period, and still at 95° C., each tube received 10μl of a mixture comprising 2.25 μl of Cleavase® A/G nuclease extract(prepared as described in Example 2) and 5 pmole of the probeoligonucleotide (SEQ ID NO:43), in 20 mM MOPS, pH 7.5 with 0.1% each ofTween 20 and NP-40, 4 mM MnCl₂ and 100 mM KCl. The reactions werebrought to 62° C. for 15 minutes and stopped by the addition of 12 μl of95% formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heatedto 75° C. for 2 minutes immediately before electrophoresis through a 20%acrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA. The products of the reactions werevisualized by the use of an Hitachi FMBIO fluorescence imager. Theresults are displayed in FIG. 35.

In FIG. 35, lane 1 contains the products of the reaction containing theprobe (SEQ ID NO:43), the invader oligonucleotide (SEQ ID NO:46) andhuman genomic DNA. Examination of lane 1 shows that the probe andinvader oligonucleotides are specific for the target sequence, and thatthe presence of genomic DNA does not cause any significant backgroundcleavage.

In FIG. 35, lanes 2 and 3 contain reaction products from reactionscontaining the single-stranded target DNA (M13mp18), the probe (SEQ IDNO:43) and the invader oligonucleotide (SEQ ID NO:46) in the absence orpresence of human genomic DNA, respectively. Examination of lanes 2 and3 demonstrate that the invader detection assay may be used to detect thepresence of a specific sequence on a single-stranded target molecule inthe presence or absence of a large excess of competitor DNA (humangenomic DNA).

In FIG. 35, lanes 4 and 5 contain reaction products from reactionscontaining the double-stranded target DNA (M13mp19), the probe (SEQ IDNO:43) and the invader oligonucleotide (SEQ ID NO:46) in the absence orpresence of human genomic DNA, respectively. Examination of lanes 4 and5 show that double stranded target molecules are eminently suitable forinvader-directed detection reactions. The success of this reaction usinga short duplexed molecule, M13mp19, as the target in a background of alarge excess of genomic DNA is especially noteworthy as it would beanticipated that the shorter and less complex M13 DNA strands would beexpected to find their complementary strand more easily than would thestrands of the more complex human genomic DNA. If the M13 DNA reannealedbefore the probe and/or invader oligonucleotides could bind to thetarget sequences along the M13 DNA, the cleavage reaction would beprevented. In addition, because the denatured genomic DNA wouldpotentially contain regions complementary to the probe and/or invaderoligonucleotides it was possible that the presence of the genomic DNAwould inhibit the reaction by binding these oligonucleotides therebypreventing their hybridization to the M13 target. The above resultsdemonstrate that these theoretical concerns are not a problem under thereaction conditions employed above.

In addition to demonstrating that the invader detection assay may beused to detect sequences present in a double-stranded target, these dataalso show that the presence of a large amount of non-target DNA (470ng/20 μl reaction) does not lessen the specificity of the cleavage.While this amount of DNA does show some impact on the rate of productaccumulation, probably by binding a portion of the enzyme, the nature ofthe target sequence, whether single- or double-stranded nucleic acid,does not limit the application of this assay.

EXAMPLE 15 Signal Accumulation in the Invader-Directed Cleavage Assay asa Function of Target Concentration

To investigate whether the invader-directed cleavage assay could be usedto indicate the amount of target nucleic acid in a sample, the followingexperiment was performed. Cleavage reactions were assembled whichcontained an invader oligonucleotide (SEQ ID NO:46), a labelled probe(SEQ ID NO:43) and a target nucleic acid, M13mp19. A series ofreactions, which contained smaller and smaller amounts of the M13 targetDNA, was employed in order to examine whether the cleavage productswould accumulate in a manner that reflected the amount of target DNApresent in the reaction.

The reactions were conducted as follows. A master mix containing enzymeand buffer was assembled. Each 5 μl of the master mixture contained 25ng of Cleavase® BN nuclease in 20 mM MOPS (pH 7.5) with 0.1% each ofTween 20 and NP-40, 4 mM MnCl₂ and 100 mM KCl. For each of the cleavagereactions shown in lanes 4-13 of FIG. 36, a DNA mixture was generatedwhich contained 5 pmoles of the fluorescein-labelled probeoligonucleotide (SEQ ID NO:43), 50 pmoles of the invader oligonucleotide(SEQ ID NO:46) and 100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.01 or 0.005fmoles of single-stranded M13mp19, respectively, for every 5 μl of theDNA mixture. The DNA solutions were covered with a drop of ChillOut®evaporation barrier (MJ Research) and brought to 61° C. The cleavagereactions were started by the addition of 5 μl of the enzyme mixture toeach of tubes (final reaction volume was 10 μl). After 30 minutes at 61°C., the reactions were terminated by the addition of 8 μl of 95%formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to90° C. for 1 minutes immediately before electrophoresis through a 20%denaturing acrylamide gel (19:1 cross-linked) with 7 M urea, in a buffercontaining 45 mM Tris-Borate (pH 8.3), 1.4 mM EDTA. To provide reference(i.e., standards), 1.0, 0.1 and 0.01 pmole aliqouts offluorescein-labelled probe oligonucleotide (SEQ ID NO:43) were dilutedwith the above formamide solution to a final volume of 18 μl. Thesereference markers were loaded into lanes 1-3, respectively of the gel.The products of the cleavage reactions (as well as the referencestandards) were visualized following electrophoresis by the use of aHitachi FMBIO fluorescence imager. The results are displayed in FIG. 36.

In FIG. 36, boxes appear around fluorescein-containing nucleic acid(i.e., the cleaved and uncleaved probe molecules) and the amount offluorescein contained within each box is indicated under the box. Thebackground fluorescence of the gel (see box labelled “background”) wassubtracted by the fluoro-imager to generate each value displayed under abox containing cleaved or uncleaved probe products (the boxes arenumbered 1-14 at top left with a V followed by a number below the box).The lane marked “M” contains fluoresceinated oligonucleotides whichserved as markers.

The results shown in FIG. 36, demonstrate that the accumulation ofcleaved probe molecules in a fixed-length incubation period reflects theamount of target DNA present in the reaction. The results alsodemonstrate that the cleaved probe products accumulate in excess of thecopy number of the target. This is clearly demonstrated by comparing theresults shown in lane 3, in which 10 fmole (0.01 pmole) of uncut probeare displayed with the results shown in 5, where the products whichaccumulated in response to the presence of 10 fmole of target DNA aredisplayed. These results show that the reaction can cleave hundreds ofprobe oligonucleotide molecules for each target molecule present,dramatically amplifying the target-specific signal generated in theinvader-directed cleavage reaction.

EXAMPLE 16 Effect of Saliva Extract on the Invader-Directed CleavageAssay

For a nucleic acid detection method to be useful in a medical (i.e., adiagnostic) setting, it must not be inhibited by materials andcontaminants likely to be found in a typical clinical specimen. To testthe susceptibility of the invader-directed cleavage assay to variousmaterials, including but not limited to nucleic acids, glycoproteins andcarbohydrates, likely to be found in a clinical sample, a sample ofhuman saliva was prepared in a manner consistent with practices in theclinical laboratory and the resulting saliva extract was added to theinvader-directed cleavage assay. The effect of the saliva extract uponthe inhibition of cleavage and upon the specificity of the cleavagereaction was examined.

One and one-half milliliters of human saliva were collected andextracted once with an equal volume of a mixture containingphenol:chloroform:isoamyl alcohol (25:24:1). The resulting mixture wascentrifuged in a microcentrifuge to separate the aqueous and organicphases. The upper, aqueous phase was transferred to a fresh tube.One-tenth volumes of 3 M NaOAc were added and the contents of the tubewere mixed. Two volumes of 100% ethyl alcohol were added to the mixtureand the sample was mixed and incubated at room temperature for 15minutes to allow a precipitate to form. The sample was centrifuged in amicrocentrifuge at 13,000 rpm for 5 minutes and the supernatant wasremoved and discarded. A milky pellet was easily visible. The pellet wasrinsed once with 70% ethanol, dried under vacuum and dissolved in 200 μlof 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA (this constitutes the salivaextract). Each μl of the saliva extract was equivalent to 7.5 μl ofsaliva. Analysis of the saliva extract by scanning ultravioletspectrophotometry showed a peak absorbance at about 260 nm and indicatedthe presence of approximately 45 ng of total nucleic acid per μl ofextract.

The effect of the presence of saliva extract upon the following enzymeswas examined: Cleavase® BN nuclease, Cleavase® A/G nuclease and threedifferent lots of DNAPTaq: AmpliTaq® (Perkin Elmer; a recombinant formof DNAPTaq), AmpliTaq® LD (Perkin-Elmer; a recombinant DNAPTaqpreparation containing very low levels of DNA) and Taq DNA polymerase(Fischer). For each enzyme tested, an enzyme/probe mixture was madecomprising the chosen amount of enzyme with 5 pmole of the probeoligonucleotide (SEQ ID NO:43) in 10 μl of 20 mM MOPS (pH 7.5)containing 0.1% each of Tween 20 and NP-40, 4 mM MnCl₂, 100 mM KCl and100 μg/ml BSA. The following amounts of enzyme were used: 25 ng ofCleavase® BN prepared as described in Example 9; 2 μl of Cleavase® A/Gnuclease extract prepared as described in Example 2; 2.25 μl (11.25polymerase units) the following DNA polymerases: AmpliTaq® DNApolymerase (Perkin Elmer); AmpliTaq® DNA polymerase LD (low DNA; fromPerkin Elmer); Taq DNA polymerase (Fisher Scientific).

For each of the reactions shown in FIG. 37, except for that shown inlane 1, the target DNA (50 fmoles of single-stranded M13mp19 DNA) wascombined with 50 pmole of the invader oligonucleotide (SEQ ID NO:46) and5 pmole of the probe oligonucleotide (SEQ ID NO:43); target DNA wasomitted in reaction 1 (lane 1). Reactions 1, 3, 5, 7, 9 and 11 included1.5 μl of saliva extract. These mixtures were brought to a volume of 5μl with distilled water, overlaid with a drop of ChillOut® evaporationbarrier (MJ Research) and brought to 95° C. for 10 minutes. The cleavagereactions were then started by the addition of 5 μl of the desiredenzyme/probe mixture; reactions 1, 4 and 5 received Cleavase® A/Gnuclease. Reactions 2 and 3 received Cleavase® BN; reactions 6 and 7received AmpliTaq®; reactions 8 and 9 received AmoliTaq® LD; andreactions 10 and 11 received Taq DNA Polymerase from Fisher Scientific.

The reactions were incubated at 63° C. for 30 minutes and were stoppedby the addition of 6 μl of 95% formamide with 20 mM EDTA and 0.05%marker dyes. Samples were heated to 75° C. for 2 minutes immediatelybefore electrophoresis through a 20% acrylamide gel (19:1 cross-linked),with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.The products of the reactions were visualized by the use of an HitachiFMBIO fluorescence imager, and the results are displayed in FIG. 37.

A pairwise comparison of the lanes shown in FIG. 37 without and with thesaliva extract, treated with each of the enzymes, shows that the salivaextract has different effects on each of the enzymes. While theCleavase® BN nuclease and the AmpliTaq® are significantly inhibited fromcleaving in these conditions, the Cleavase® A/G nuclease and AmpliTaq®LD display little difference in the yield of cleaved probe. Thepreparation of Taq DNA polymerase from Fisher Scientific shows anintermediate response, with a partial reduction in the yield of cleavedproduct. From the standpoint of polymerization, the three DNAPTaqvariants should be equivalent; these should be the same protein with thesame amount of synthetic activity. It is possible that the differencesobserved could be due to variations in the amount of nuclease activitypresent in each preparation caused by different handling duringpurification, or by different purification protocols. In any case,quality control assays designed to assess polymerization activity incommercial DNAP preparations would be unlikely to reveal variation inthe amount of nuclease activity present. If preparations of DNAPTaq werescreened for full 5′ nuclease activity (i.e., f the 5′ nuclease activitywas specifically quantitated), it is likely that the preparations woulddisplay sensitivities (to saliva extract) more in line with thatobserved using Cleavase® A/G nuclease, from which DNAPTaq differs by avery few amino acids.

It is worthy of note that even in the slowed reactions of Cleavase® BNand the DNAPTaq variants there is no noticeable increase in non-specificcleavage of the probe oligonucleotide due to inappropriate hybridizationor saliva-borne nucleases.

EXAMPLE 17 Comparison of Additional 5′ Nucleases in the Invader-DirectedCleavage Assay

A number of eubacterial Type A DNA polymerases (i.e., Pol I type DNApolymerases) have been shown to function as structure specificendonucleases (Example 1 and Lyamichev et al., supra). In this example,it was demonstrated that the enzymes of this class can also be made tocatalyze the invader-directed cleavage of the present invention, albeitnot as efficiently as the Cleavase® enzymes.

Cleavase® BN nuclease and Cleavase® A/G nuclease were tested along sidethree different thermostable DNA polymerases: Thermus aquaticus DNApolymerase (Promega), Thermus thermophilus and Thermus flavus DNApolymerases (Epicentre). The enzyme mixtures used in the reactions shownin lanes 1-11 of FIG. 38 contained the following, each in a volume of 5μl: Lane 1: 20 mM MOPS (pH 7.5) with 0.1% each of Tween 20 and NP-40, 4mM MnCl₂, 100 mM KCl; Lane 2: 25 ng of Cleavase® BN nuclease in the samesolution described for lane 1; Lane 3: 2.25 μl of Cleavase® A/G nucleaseextract (prepared as described in Example 2), in the same solutiondescribed for lane 1; Lane 4: 2.25 μl of Cleavase® A/G nuclease extractin 20 mM Tris-Cl, (pH 8.5), 4 mM MgCl₂ and 100 mM KCl; Lane 5: 11.25polymerase units of Taq DNA polymerase in the same buffer described forlane 4; Lane 6: 11.25 polymerase units of Tth DNA polymerase in the samebuffer described for lane 1; Lane 7: 11.25 polymerase units of Tth DNApolymerase in a 2× concentration of the buffer supplied by themanufacturer, supplemented with 4 mM MnCl₂; Lane 8: 11.25 polymeraseunits of Tth DNA polymerase in a 2× concentration of the buffer suppliedby the manufacturer, supplemented with 4 mM MgCl₂; Lane 9: 2.25polymerase units of Tfl DNA polymerase in the same buffer described forlane 1; Lane 10: 2.25 polymerase units of Tfl polymerase in a 2×concentration of the buffer supplied by the manufacturer, supplementedwith 4 mM MnCl₂; Lane 11: 2.25 polymerase units of Tfl DNA polymerase ina 2× concentration of the buffer supplied by the manufacturer,supplemented with 4 mM MgCl₂.

Sufficient target DNA, probe and invader for all 11 reactions wascombined into a master mix. This mix contained 550 fmoles ofsingle-stranded M13mp19 target DNA, 550 pmoles of the invaderoligonucleotide (SEQ ID NO:46) and 55 pmoles of the probeoligonucleotide (SEQ ID NO:43), each as depicted in FIG. 32 c, in 55 μlof distilled water. Five μl of the DNA mixture was dispensed into eachof 11 labeled tubes and overlaid with a drop of ChillOut® evaporationbarrier (MJ Research). The reactions were brought to 63° C. and cleavagewas started by the addition of 5 μl of the appropriate enzyme mixture.The reaction mixtures were then incubated at 63° C. temperature for 15minutes. The reactions were stopped by the addition of 8 μl of 95%formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to90° C. for 1 minute immediately before electrophoresis through a 20%acrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mMTris-Borate (pH 8.3), 1.4 mM EDTA. Following electrophoresis, theproducts of the reactions were visualized by the use of an Hitachi FMBIOfluorescence imager, and the results are displayed in FIG. 38.Examination of the results shown in FIG. 38 demonstrates that all of the5′ nucleases tested have the ability to catalyze invader-directedcleavage in at least one of the buffer systems tested. Although notoptimized here, these cleavage agents are suitable for use in themethods of the present invention.

EXAMPLE 18 The Invader-Directed Cleavage Assay Can Detect Single BaseDifferences in Target Nucleic Acid Sequences

The ability of the invader-directed cleavage assay to detect single basemismatch mutations was examined. Two target nucleic acid sequencescontaining Cleavase® enzyme-resistant phosphorothioate backbones werechemically synthesized and purified by polyacrylamide gelelectrophoresis. Targets comprising phosphorothioate backbones were usedto prevent exonucleolytic nibbling of the target when duplexed with anoligonucleotide. A target oligonucleotide, which provides a targetsequence that is completely complementary to the invader oligonucleotide(SEQ ID NO:46) and the probe oligonucleotide (SEQ ID NO:43), containedthe following sequence: 5′-CCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGC-3′(SEQ ID NO:47). A second target sequence containing a single base changerelative to SEQ ID NO:47 was synthesized:5′-CCTTTCGCTCTCTTCCCTTCCTTTCTCGCC ACGTTCGCCGGC-3 (SEQ ID NO:48; thesingle base change relative to SEQ ID NO:47 is shown using bold andunderlined type). The consequent mismatch occurs within the “Z” regionof the target as represented in FIG. 29.

To discriminate between two target sequences which differ by thepresence of a single mismatch), invader-directed cleavage reactions wereconducted using two different reaction temperatures (55° C. and 60° C.).Mixtures containing 200 fmoles of either SEQ ID NO:47 or SEQ ID NO:48, 3pmoles of fluorescein-labelled probe oligonucleotide (SEQ ID NO:43), 7.7pmoles of invader oligonucleotide (SEQ ID NO:46) and 2 μl of Cleavase®A/G nuclease extract (prepared as described in Example 2) in 9 μl of 10mM MOPS (pH 7.4) with 50 mM KCl were assembled, covered with a drop ofChillOut® evaporation barrier (MJ Research) and brought to theappropriate reaction temperature. The cleavage reactions were initiatedby the addition of 1 μl of 20 mM MgCl₂. After 30 minutes at either 55°C. or 60° C., 10 μl of 95% formamide with 20 mM EDTA and 0.05% markerdyes was added to stop the reactions. The reaction mixtures where thenheated to 90° C. for one minute prior to loading 4 μl onto 20%denaturing polyacrylamide gels. The resolved reaction products werevisualized using a Hitachi FMBIO fluorescence imager. The resultingimage is shown in FIG. 39.

In FIG. 39, lanes 1 and 2 show the products from reactions conducted at55° C.; lanes 3 and 4 show the products from reactions conducted at 60°C. Lanes 1 and 3 contained products from reactions containing SEQ IDNO:47 (perfect match to probe) as the target. Lanes 2 and 4 containedproducts from reactions containing SEQ ID NO:48 (single base mis-matchwith probe) as the target. The target that does not have a perfecthybridization match (i.e., complete complementarity) with the probe willnot bind as strongly, i.e., the T_(m) of that duplex will be lower thanthe T_(m) of the same region if perfectly matched. The results presentedhere show that reaction conditions can be varied to either accommodatethe mis-match (e.g., by lowering the temperature of the reaction) or toexclude the binding of the mis-matched sequence (e.g., by raising thereaction temperature).

The results shown in FIG. 39 demonstrate that the specific cleavageevent which occurs in invader-directed cleavage reactions can beeliminated by the presence of a single base mis-match between the probeoligonucleotide and the target sequence. Thus, reaction conditions canbe chosen so as to exclude the hybridization of mis-matchedinvader-directed cleavage probes thereby diminishing or even eliminatingthe cleavage of the probe. In an extension of this assay system,multiple cleavage probes, each possessing a separate reporter molecule(i.e., a unique label), could also be used in a single cleavagereaction, to simultaneously probe for two or more variants in the sametarget region. The products of such a reaction would allow not only thedetection of mutations which exist within a target molecule, but wouldalso allow a determination of the relative concentrations of eachsequence (i.e., mutant and wild type or multiple different mutants)present within samples containing a mixture of target sequences. Whenprovided in equal amounts, but in a vast excess (e.g. at least a100-fold molar excess; typically at least 1 pmole of each probeoligonucleotide would be used when the target sequence was present atabout 10 fmoles or less) over the target and used in optimizedconditions. As discussed above, any differences in the relative amountsof the target variants will not affect the kinetics of hybridization, sothe amounts of cleavage of each probe will reflect the relative amountsof each variant present in the reaction.

The results shown in the example clearly demonstrate that theinvader-directed cleavage reaction can be used to detect single basedifference between target nucleic acids.

EXAMPLE 19 The Invader-Directed Cleavage Reaction is Insensitive toLarge Changes in Reaction Conditions

The results shown above demonstrated that the invader-directed cleavagereaction can be used for the detection of target nucleic acid sequencesand that this assay can be used to detect single base difference betweentarget nucleic acids. These results demonstrated that 5′ nucleases(e.g., Cleavase®BN, Cleavase® A/G, DNAPTaq, DNAPTth, DNAPTfl) could beused in conjunction with a pair of overlapping oligonucleotides as anefficient way to recognize nucleic acid targets. In the experimentsbelow it is demonstrated that invasive cleavage reaction is relativelyinsensitive to large changes in conditions thereby making the methodsuitable for practice in clinical laboratories.

The effects of varying the conditions of the cleavage reaction wereexamined for their effect(s) on the specificity of the invasive cleavageand the on the amount of signal accumulated in the course of thereaction. To compare variations in the cleavage reaction a “standard”invader cleavage reaction was first defined. In each instance, unlessspecifically stated to be otherwise, the indicated parameter of thereaction was varied, while the invariant aspects of a particular testwere those of this standard reaction. The results of these tests areshown in FIGS. 42-51.

-   -   a) The Standard Invader-Directed Cleavage Reaction

The standard reaction was defined as comprising 1 fmole of M13mp18single-stranded target DNA (New England Biolabs), 5 pmoles of thelabeled probe oligonucleotide (SEQ ID NO:49), 10 pmole of the upstreaminvader oligonucleotide (SEQ ID NO:50) and 2 units of Cleavase® A/G in10 μl of 10 mM MOPS, pH 7.5 with 100 mM KCl, 4 mM MnCl₂, and 0.05% eachTween-20 and Nonidet-P40. For each reaction, the buffers, salts andenzyme were combined in a volume of 5 μl; the DNAs (target and twooligonucleotides) were combined in 5 μl of dH₂O and overlaid with a dropof ChillOut® evaporation barrier (MJ Research). When multiple reactionswere performed with the same reaction constituents, these formulationswere expanded proportionally.

Unless otherwise stated, the sample tubes with the DNA mixtures werewarmed to 61° C., and the reactions were started by the addition of 5 μlof the enzyme mixture. After 20 minutes at this temperature, thereactions were stopped by the addition of 8 μl of 95% formamide with 20mM EDTA and 0.05% marker dyes. Samples were heated to 75° C. for 2minutes immediately before electrophoresis through a 20% acrylamide gel(19:1 cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH8.3, 1.4 mM EDTA. The products of the reactions were visualized by theuse of an Hitachi FMBIO fluorescence imager. In each case, the uncutprobe material was visible as an intense black band or blob, usually inthe top half of the panel, while the desired products of invaderspecific cleavage were visible as one or two narrower black bands,usually in the bottom half of the panel. Under some reaction conditions,particulary those with elevated salt concentrations, a secondarycleavage product is also visible (thus generating a doublet). Ladders oflighter grey bands generally indicate either exonuclease nibbling of theprobe oligonucleotide or heat-induced, non-specific breakage of theprobe.

FIG. 41 depicts the annealing of the probe and invader oligonucleotidesto regions along the M13mp18 target molecule (the bottom strand). InFIG. 41 only a 52 nucleotide portion of the M13mp18 molecule is shown;this 52 nucleotide sequence is listed in SEQ ID NO:42 (this sequence isidentical in both M13mp18 and M13mp19). The probe oligonucleotide (topstrand) contains a Cy3 amidite label at the 5′ end; the sequence of theprobe is 5′-AGAAAGGAAGGGAAGAAAGCGAAA GGT-3′ (SEQ ID NO:49. The bold typeindicates the presence of a modified base (2′-O—CH₃). Cy3 amidite(Pharmacia) is a indodicarbocyanine dye amidite which can beincorporated at any position during the synthesis of oligonucleotides;Cy3 μfluoresces in the yellow region (excitation and emission maximum of554 and 568 nm, respectively). The invader oligonucleotide (middlestrand) has the following sequence: 5′-GCCGGCGAACGTGGCGAGAAAGGA-3′ (SEQID NO:50).

-   -   b) KCl Titration

FIG. 42 shows the results of varying the KCl concentration incombination with the use of 2 mM MnCl₂, in an otherwise standardreaction. The reactions were performed in duplicate for confirmation ofobservations; the reactions shown in lanes 1 and 2 contained no addedKCl, lanes 3 and 4 contained KCl at 5 mM, lanes 5 and 6 contained 25 mMKCl, lanes 7 and 8 contained 50 mM KCl, lanes 9 and 10 contained 100 mMKCl and lanes 11 and 12 contained 200 mM KCl. These results show thatthe inclusion of KCl allows the generation of a specific cleavageproduct. While the strongest signal is observed at the 100 mM KClconcentration, the specificity of signal in the other reactions with KClat or above 25 mM indicates that concentrations in the full range (i.e.,25-200 mM) may be chosen if it is so desirable for any particularreaction conditions.

As shown in FIG. 42, the invader-directed cleavage reaction requires thepresence of salt (e.g., KCl) for effective cleavage to occur. In otherreactions, it has been found that KCl can inhibit the activity ofcertain Cleavase® enzymes when present at concentrations above about 25mM (For example, in cleavage reactions using the S-60 oligonucleotideshown in FIG. 30, in the absence of primer, the Cleavase® BN enzymeloses approximately 50% of its activity in 50 mM KCl). Therefore, theuse of alternative salts in the invader-directed cleavage reaction wasexamined. In these experiments, the potassium ion was replaced witheither Na⁺ or Li⁺ or the chloride ion was replaced with glutamic acid.The replacement of KCl with alternative salts is described below insections c-e.

-   -   c) NaCl Titration

FIG. 43 shows the results of using various concentrations of NaCl inplace of KCl (lanes 3-10) in combination with the use 2 mM MnCl₂, in anotherwise standard reaction, in comparison to the effects seen with 100mM KCl (lanes 1 and 2). The reactions analyzed in lanes 3 and 4contained NaCl at 75 mM, lanes 5 and 6 contained 100 mM, lanes 7 and 8contained 150 mM and lanes 9 and 10 contained 200 mM. These results showthat NaCl can be used as a replacement for KCl in the invader-directedcleavage reaction (i.e., the presence of NaCl, like KCl, enhancesproduct accumulation).

-   -   d) LiCl Titration

FIG. 44 shows the results of using various concentrations of LiCl inplace of KCl (lanes 3-14) in otherwise standard reactions, compared tothe effects seen with 100 mM KCl (lanes 1 and 2). The reactions analyzedin lanes 3 and 4 contained LiCl at 25 mM, lanes 5 and 6 contained 50 mM,lanes 7 and 8 contained 75 mM, lanes 9 and 10 contained 100 mM, lanes 11and 12 contained 150 mM and lanes 13 and 14 contained 200 mM. Theseresults demonstrate that LiCl can be used as a suitable replacement forKCl in the invader-directed cleavage reaction (i.e., the presence ofLiCl, like KCl, enhances product accumulation).

-   -   e) KGlu Titration

FIG. 45 shows the results of using a glutamate salt of potassium (KGlu)in place of the more commonly used chloride salt (KCl) in reactionsperformed over a range of temperatures. KGlu has been shown to be ahighly effective salt source for some enzymatic reactions, showing abroader range of concentrations which permit maximum enzymatic activity[Leirmo et al. (1987) Biochem. 26:2095]. The ability of KGlu tofacilitate the annealing of the probe and invader oligonucleotides tothe target nucleic acid was compared to that of LiCl. In theseexperiments, the reactions were run for 15 minutes, rather than thestandard 20 minutes. The reaction analyzed in lane 1 contained 150 mMLiCl and was run at 65° C.; the reactions analyzed in lanes 2-4contained 200 mM, 300 mM and 400 mM KGlu, respectively and were run at65° C. The reactions analyzed in lanes 5-8 repeated the array of saltconcentrations used in lanes 1-4, but were performed at 67° C.; lanes9-12 show the same array run at 69° C. and lanes 13-16 show the samearray run at 71° C. The results shown in FIG. 45 demonstrate that KGluwas very effective as a salt in the invasive cleavage reactions. Inaddition, these data show that the range of allowable KGluconcentrations was much greater than that of LiCl, with full activityapparent even at 400 mM KGlu.

-   -   f) MnCl₂ and MgCl₂ Titration and Ability to Replace MnCl₂ with        MgCl₂

In some instances it may be desirable to perform the invasive cleavagereaction in the presence of Mg²⁺, either in addition to, or in place ofMn²⁺ as the necessary divalent cation required for activity of theenzyme employed. For example, some common methods of preparing DNA frombacterial cultures or tissues use MgCl₂ in solutions which are used tofacilitate the collection of DNA by precipitation. In addition, elevatedconcentrations (i.e., greater than 5 mM) of divalent cation can be usedto facilitate hybridization of nucleic acids, in the same way that themonovalent salts were used above, thereby enhancing the invasivecleavage reaction. In this experiment, the tolerance of the invasivecleavage reaction was examined for 1) the substitution of MgCl₂ forMnCl₂ and for the ability to produce specific product in the presence ofincreasing concentrations of MgCl₂ and MnCl₂.

FIG. 46 shows the results of either varying the concentration of MnCl₂from 2 mM to 8 mM, replacing the MnCl₂ with MgCl₂ at 2 to 4 mM, or ofusing these components in combination in an otherwise standard reaction.The reactions analyzed in lanes 1 and 2 contained 2 mM each MnCl₂ andMgCl₂, lanes 3 and 4 contained 2 mM MnCl₂ only, lanes 5 and 6 contained3 mM MnCl₂, lanes 7 and 8 contained 4 mM MnCl₂, lanes 9 and 10 contained8 mM MnCl₂. The reactions analyzed in lanes 11 and 12 contained 2 mMMgCl₂ and lanes 13 and 14 contained 4 mM MgCl₂. These results show thatboth MnCl₂ and MgCl₂ can be used as the necessary divalent cation toenable the cleavage activity of the Cleavase® A/G enzyme in thesereactions and that the invasive cleavage reaction can tolerate a broadrange of concentrations of these components.

In addition to examining the effects of the salt environment on the rateof product accumulation in the invasive cleavage reaction, the use ofreaction constituents shown to be effective in enhancing nucleic acidhybridization in either standard hybridization assays (e.g., blothybridization) or in ligation reactions was examined. These componentsmay act as volume excluders, increasing the effective concentration ofthe nucleic acids of interest and thereby enhancing hybridization, orthey may act as charge-shielding agents to minimize repulsion betweenthe highly charged backbones of the nucleic acids strands. The resultsof these experiments are described in sections g and h below.

-   -   g) Effect of CTAB Addition

The polycationic detergent cetyltrietheylammonium bromide (CTAB) hasbeen shown to dramatically enhance hybridization of nucleic acids[Pontius and Berg (1991) Proc. Natl. Acad. Sci. USA 88:8237]. The datashown in FIG. 47 depicts the results of adding the detergent CTAB toinvasive cleavage reactions in which 150 mM LiCl was used in place ofthe KCl in otherwise standard reactions. Lane 1 shows unreacted (i.e.,uncut) probe, and the reaction shown in lane 1 is the LiCl-modifiedstandard reaction without CTAB. The reactions analyzed in lanes 3 and 4contained 100 μM CTAB, lanes 5 and 6 contained 200 μM CTAB, lanes 7 and8 contained 400 μM CTAB, lanes 9 and 10 contained 600 μM CTAB, lanes 11and 12 contained 800 μM CTAB and lanes 13 and 14 contained 1 mM CTAB.These results showed that the lower amounts of CTAB may have a verymoderate enhancing effect under these reaction conditions, and thepresence of CTAB in excess of about 500 μM was inhibitory to theaccumulation of specific cleavage product.

-   -   h) Effect of PEG Addition

FIG. 48 shows the effect of adding polyethylene glycol (PEG) at variouspercentage (w/v) concentrations to otherwise standard reactions. Theeffects of increasing the reaction temperature of the PEG-containingreactions was also examined. The reactions assayed in lanes 1 and 2 werethe standard conditions without PEG, lanes 3 and 4 contained 4% PEG,lanes 5 and 6 contained 8% PEG and lanes 7 and 8 contained 12% PEG. Eachof the aforementioned reactions was performed at 61° C. The reactionsanalyzed in lanes 9, 10, 11 and 12 were performed at 65 ° C., andcontained 0%, 4%, 8% and 12% PEG, respectively. These results show thatat all percentages tested, and at both temperatures tested, theinclusion of PEG substantially eliminated the production of specificcleavage product.

In addition to the data presented above (i.e., effect of CTAB and PEGaddition), the presence of 1× Denhardts in the reaction mixture wasfound to have no adverse effect upon the cleavage reaction [50×Denhardt's contains per 500 ml: 5 g Ficoll, 5 g polyvinylpyrrolidone, 5g BSA]. In addition, the presence of each component of Denhardt's wasexamined individually (i.e., Ficoll alone, polyvinylpyrrolidone alone,BSA alone) for the effect upon the invader-directed cleavage reaction;no adverse effect was observed.

-   -   i) Effect of the Addition of Stabilizing Agents

Another approach to enhancing the output of the invasive cleavagereaction is to enhance the activity of the enzyme employed, either byincreasing its stability in the reaction environment or by increasingits turnover rate. Without regard to the precise mechanism by whichvarious agents operate in the invasive cleavage reaction, a number ofagents commonly used to stabilize enzymes during prolonged storage weretested for the ability to enhance the accumulation of specific cleavageproduct in the invasive cleavage reaction.

FIG. 49 shows the effects of adding glycerol at 15% and of adding thedetergents Tween-20 and Nonidet-P40 at 1.5%, alone or in combination, inotherwise standard reactions. The reaction analyzed in lane 1 was astandard reaction. The reaction analyzed in lane 2 contained 1.5% NP-40,lane 3 contained 1.5% Tween 20, lane 4 contained 15% glycerol. Thereaction analyzed in lane 5 contained both Tween-20 and NP-40 added atthe above concentrations, lane 6 contained both glycerol and NP-40, lane7 contained both glycerol and Tween-20, and lane 8 contained all threeagents. The results shown in FIG. 49 demonstrate that under theseconditions these adducts had little or no effect on the accumulation ofspecific cleavage product.

FIG. 50 shows the effects of adding gelatin to reactions in which thesalt identity and concentration were varied from the standard reaction.In addition, all of these reactions were performed at 65° C., instead of61° C. The reactions assayed in lanes 1-4 lacked added KCl, and included0.02%, 0.05%, 0.1% or 0.2% gelatin, respectively. Lanes 5, 6, 7 and 8contained the same titration of gelatin, respectively, and included 100mM KCl. Lanes 9, 10, 11 and 12, also had the same titration of gelatin,and additionally included 150 mM LiCl in place of KCl. Lanes 13 and 14show reactions that did not include gelatin, but which contained either100 mM KCl or 150 mM LiCl, respectively. The results shown in FIG. 50demonstrated that in the absence of salt the gelatin had a moderatelyenhancing effect on the accumulation of specific cleavage product, butwhen either salt (KCl or LiCl) was added to reactions performed underthese conditions, increasing amounts of gelatin reduced the productaccumulation.

-   -   j) Effect of Adding Large Amounts of Non-Target Nucleic Acid

In detecting specific nucleic acid sequences within samples, it isimportant to determine if the presence of additional genetic material(i.e., non-target nucleic acids) will have a negative effect on thespecificity of the assay. In this experiment, the effect of includinglarge amounts of non-target nucleic acid, either DNA or RNA, on thespecificity of the invasive cleavage reaction was examined. The data wasexamined for either an alteration in the expected site of cleavage, orfor an increase in the nonspecific degradation of the probeoligonucleotide.

FIG. 51 shows the effects of adding non-target nucleic acid (e.g.,genomic DNA or tRNA) to an invasive cleavage reaction performed at 65°C., with 150 mM LiCl in place of the KCl in the standard reaction. Thereactions assayed in lanes 1 and 2 contained 235 and 470 ng of genomicDNA, respectively. The reactions analyzed in lanes 3, 4, 5 and 6contained 100 ng, 200 ng, 500 ng and 1 μg of tRNA, respectively. Lane 7represents a control reaction which contained no added nucleic acidbeyond the amounts used in the standard reaction. The results shown inFIG. 51 demonstrate that the inclusion of non-target nucleic acid inlarge amounts could visibly slow the accumulation of specific cleavageproduct (while not limiting the invention to any particular mechanism,it is thought that the additional nucleic acid competes for binding ofthe enzyme with the specific reaction components). In additionalexperiments it was found that the effect of adding large amounts ofnon-target nucleic acid can be compensated for by increasing the enzymein the reaction. The data shown in FIG. 51 also demonstrate that a keyfeature of the invasive cleavage reaction, the specificity of thedetection, was not compromised by the presence of large amounts ofnon-target nucleic acid.

In addition to the data presented above, invasive cleavage reactionswere run with succinate buffer at pH 5.9 in place of the MOPS bufferused in the “standard” reaction; no adverse effects were observed.

The data shown in FIGS. 42-51 and described above demonstrate that theinvasive cleavage reaction can be performed using a wide variety ofreaction conditions and is therefore suitable for practice in clinicallaboratories.

EXAMPLE 20 Detection of RNA Targets by Invader-Directed Cleavage

In addition to the clinical need to detect specific DNA sequences forinfectious and genetic diseases, there is a need for technologies thatcan quantitatively detect target nucleic acids that are composed of RNA.For example, a number of viral agents, such as hepatitis C virus (HCV)and human immunodeficiency virus (HIV) have RNA genomic material, thequantitative detection of which can be used as a measure of viral loadin a patient sample. Such information can be of critical diagnostic orprognostic value.

Hepatitis C virus (HCV) infection is the predominant cause ofpost-transfusion non-A, non-B (NANB) hepatitis around the world. Inaddition, HCV is the major etiologic agent of hepatocellular carcinoma(HCC) and chronic liver disease world wide. The genome of HCV is a small(9.4 kb) RNA molecule. In studies of transmission of HCV by bloodtransfusion it has been found the presence of HCV antibody, as measuredin standard immunological tests, does not always correlate with theinfectivity of the sample, while the presence of HCV RNA in a bloodsample strongly correlates with infectivity. Conversely, serologicaltests may remain negative in immunosuppressed infected individuals,while HCV RNA may be easily detected [J. A. Cuthbert (1994) Clin.Microbiol. Rev. 7:505].

The need for and the value of developing a probe-based assay for thedetection the HCV RNA is clear. The polymerase chain reaction has beenused to detect HCV in clinical samples, but the problems associated withcarry-over contamination of samples has been a concern. Direct detectionof the viral RNA without the need to perform either reversetranscription or amplification would allow the elimination of several ofthe points at which existing assays may fail.

The genome of the positive-stranded RNA hepatitis C virus comprisesseveral regions including 5′ and 3′ noncoding regions (i.e., 5′ and 3′untranslated regions) and a polyprotein coding region which encodes thecore protein (C), two envelope glycoproteins (E1 and E2/NS1) and sixnonstructural glycoproteins (NS2-NS5b). Molecular biological analysis ofthe HCV genome has showed that some regions of the genome are veryhighly conserved between isolates, while other regions are fairlyrapidly changeable. The 5′ noncoding region (NCR) is the most highlyconserved region in the HCV. These analyses have allowed these virusesto be divided into six basic genotype groups, and then furtherclassified into over a dozen sub-types [the nomenclature and division ofHCV genotypes is evolving; see Altamirano et al., J. Infect. Dis.171:1034 (1995) for a recent classification scheme].

In order to develop a rapid and accurate method of detecting HCV presentin infected individuals, the ability of the invader-directed cleavagereaction to detect HCV RNA was examined. Plasmids containing DNA derivedfrom the conserved 5′-untranslated region of six different HCV RNAisolates were used to generate templates for in vitro transcription. TheHCV sequences contained within these six plasmids represent genotypes 1(four sub-types represented; 1a, 1b, 1c, and Δ1c), 2, and 3. Thenomenclature of the HCV genotypes used herein is that of Simmonds et al.[as described in Altamirano et at., supra]. The Δ1c subtype was used inthe model detection reaction described below.

-   -   a) Generation of Plasmids Containing HCV Sequences

Six DNA fragments derived from HCV were generated by RT-PCR using RNAextracted from serum samples of blood donors; these PCR fragments were agift of Dr. M. Altamirano (University of British Columbia. Vancouver).These PCR fragments represent HCV sequences derived from HCV genotypes1a, 1b, 1c, Δ1c, 2c and 3a.

The RNA extraction, reverse transcription and PCR were performed usingstandard techniques (Altamirano et al., supra). Briefly, RNA wasextracted from 100 μl of serum using guanidine isothiocyanate, sodiumlauryl sarkosate and phenol-chloroform [Inchauspe et al., Hepatology14:595 (1991)]. Reverse transcription was performed according to themanufacturer's instructions using a GeneAmp rTh reverse transcriptaseRNA PCR kit (Perkin-Elmer) in the presence of an external antisenseprimer, HCV342. The sequence of the HCV342 primer is 5′-GGTTTTTCTTTGAGGTTTAG-3′ (SEQ ID NO:51). Following termination of the RT reaction, thesense primer HCV7 [5′-GCGACACTCCACCATAGAT-3′ (SEQ ID NO:52)] andmagnesium were added and a first PCR was performed. Aliquots of thefirst PCR products were used in a second (nested) PCR in the presence ofprimers HCV46 [5′-CTGTCTTCACGCAGAAAGC-3′ (SEQ ID NO:53)] and HCV308[5′-GCACGGT CTACGAGACCTC-3′ (SEQ ID NO:54)]. The PCRs produced a 281 bpproduct which corresponds to a conserved 5′ noncoding region (NCR)region of HCV between positions −284 and −4 of the HCV genome(Altramirano et al., supra).

The six 281 bp PCR fragments were used directly for cloning or they weresubjected to an additional amplification step using a 50 μl PCRcomprising approximately 100 fmoles of DNA, the HCV46 and HCV308 primersat 0.1 μM, 100 μM of all four dNTPs and 2.5 units of Taq DNA polymerasein a buffer containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂and 0.1% Tween 20. The PCRs were cycled 25 times at 96° C. for 45 sec.,55° C. for 45 sec. and 72° C. for 1 min. Two microliters of either theoriginal DNA samples or the reamplified PCR products were used forcloning in the linear pT7Blue T-vector (Novagen, Madison, Wis.)according to manufacturer's protocol. After the PCR products wereligated to the pT7Blue T-vector, the ligation reaction mixture was usedto transform competent JM109 cells (Promega). Clones containing thepT7Blue T-vector with an insert were selected by the presence ofcolonies having a white color on LB plates containing 40 μg/ml X-Gal, 40μg/ml IPTG and 50 μg/ml ampicillin. Four colonies for each PCR samplewere picked and grown overnight in 2 ml LB media containing 50 μg/mlcarbenicillin. Plasmid DNA was isolated using the following alkalineminiprep protocol. Cells from 1.5 ml of the overnight culture werecollected by centrifugation for 2 min. in a microcentrifuge (14K rpm),the supernatant was discarded and the cell pellet was resuspended in 50μl TE buffer with 10 μg/ml RNAse A (Pharmacia). One hundred microlitersof a solution containing 0.2 N NaOH, 1% SDS was added and the cells werelysed for 2 min. The lysate was gently mixed with 100 μl of 1.32 Mpotassium acetate, pH 4.8, and the mixture was centrifuged for 4 min. ina microcentrifuge (14K rpm); the pellet comprising cell debris wasdiscarded. Plasmid DNA was precipitated from the supernatant with 200 μlethanol and pelleted by centrifugation a microcentrifuge (14K rpm). TheDNA pellet was air dried for 15 min. and was then redissolved in 50 μlTE buffer (10 mM Tris-HCl, pH 7.8, 1 mM EDTA).

-   -   b) Reamplification of HCV Clones to Add the Phage T7 Promoter        for Subsequent In Vitro Transcription

To ensure that the RNA product of transcription had a discrete 3′ end itwas necessary to create linear transcription templates which stopped atthe end of the HCV sequence. These fragments were conveniently producedusing the PCR to reamplify the segment of the plasmid containing thephage promoter sequence and the HCV insert. For these studies, the cloneof HCV type Δ1c was reamplified using a primer that hybridizes to the T7promoter sequence: 5′-TAATACGACTCACTATAGGG-3′ (SEQ ID NO:55; “the T7promoter primer”) (Novagen) in combination with the 3′ terminalHCV-specific primer HCV308 (SEQ ID NO:54). For these reactions, 1 μl ofplasmid DNA (approximately 10 to 100 ng) was reamplified in a 200 μl PCRusing the T7 and HCV308 primers as described above with the exceptionthat 30 cycles of amplification were employed. The resulting ampliconwas 354 bp in length. After amplification the PCR mixture wastransferred to a fresh 1.5 ml microcentrifuge tube, the mixture wasbrought to a final concentration of 2 M NH₄OAc, and the products wereprecipitated by the addition of one volume of 100% isopropanol.Following a 10 min. incubation at room temperature, the precipitateswere collected by centrifugation, washed once with 80% ethanol and driedunder vacuum. The collected material was dissolved in 100 μlnuclease-free distilled water (Promega).

Segments of RNA were produced from this amplicon by in vitrotranscription using the RiboMAX™ Large Scale RNA Production System(Promega) in accordance with the manufacturer's instructions, using 5.3μg of the amplicon described above in a 100 μl reaction. Thetranscription reaction was incubated for 3.75 hours, after which the DNAtemplate was destroyed by the addition of 5-6 μl of RQ1 RNAse-free DNAse(1 unit/μl) according to the RiboMAX™ kit instructions. The reaction wasextracted twice with phenol/chloroform/isoamyl alcohol (50:48:2) and theaqueous phase was transferred to a fresh microcentrifuge tube. The RNAwas then collected by the addition of 10 μl of 3M NH₄OAc, pH 5.2 and 110μl of 100% isopropanol. Following a 5 min. incubation at 4° C., theprecipitate was collected by centrifugation, washed once with 80%ethanol and dried under vacuum. The sequence of the resulting RNAtranscript (HCV1.1 transcript) is listed in SEQ ID NO:56.

-   -   c) Detection of the HCV1.1 Transcript in the Invader-Directed        Cleavage Assay

If Detection of the HCV1.1 transcript was tested in the invader-directedcleavage assay using an HCV-specific probe oligonucleotide[5′-CCGGTCGTCCTGGCAAT XCC-3′ (SEQ ID NO:57); X indicates the presence ofa fluorescein dye on an a basic linker) and an HCV-specific invaderoligonucleotide [5′-GTTTATCCAAGAAAGGAC CCGGTCC-3′ (SEQ ID NO:58)] thatcauses a 6-nucleotide invasive cleavage of the probe.

Each 10 μl of reaction mixture comprised 5 pmole of the probeoligonucleotide (SEQ ID NO:57) and 10 pmole of the invaderoligonucleotide (SEQ ID NO:58) in a buffer of 10 mM MOPS, pH 7.5 with 50mM KCl, 4 mM MnCl₂, 0.05% each Tween-20 and Nonidet-P40 and 7.8 unitsRNasine ribonucleas® inhibitor (Promega). The cleavage agents employedwere Cleavase® A/G (used at 5.3 ng/10 μl reaction) or DNAPTth (used at 5polymerase units/10 μl reaction). The amount of RNA target was varied asindicated below. When RNAse treatment is indicated, the target RNAs werepre-treated with 10 μg of RNase A (Sigma) at 37° C. for 30 min. todemonstrate that the detection was specific for the RNA in the reactionand not due to the presence of any residual DNA template from thetranscription reaction. RNase-treated aliquots of the HCV RNA were useddirectly without intervening purification.

For each reaction, the target RNAs were suspended in the reactionsolutions as described above, but lacking the cleavage agent and theMnCl₂ for a final volume of 10 μl, with the invader and probe at theconcentrations listed above. The reactions were warmed to 46° C. and thereactions were started by the addition of a mixture of the appropriateenzyme with MnCl₂. After incubation for 30 min. at 46° C., the reactionswere stopped by the addition of 8 μl of 95% formamide, 10 mM EDTA and0.02% methyl violet (methyl violet loading buffer). Samples were thenresolved by electrophoresis through a 15% denaturing polyacrylamide gel(19:1 cross-linked), containing 7 M urea, in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA. Following electrophoresis, the labeledreaction products were visualized using the FMBIO-100 Image Analyzer(Hitachi), with the resulting imager scan shown in FIG. 52.

In FIG. 52, the samples analyzed in lanes 1-4 contained 1 pmole of theRNA target, the reactions shown in lanes 5-8 contained 100 fmoles of theRNA target and the reactions shown in lanes 9-12 contained 10 fmoles ofthe RNA target. All odd-numbered lanes depict reactions performed usingCleavase® A/G enzyme and all even-numbered lanes depict reactionsperformed using DNAPTth. The reactions analyzed in lanes 1, 2, 5, 6, 9and 10 contained RNA that had been pre-digested with RNase A. These datademonstrate that the invasive cleavage reaction efficiently detects RNAtargets and further, the absence of any specific cleavage signal in theRNase-treated samples confirms that the specific cleavage product seenin the other lanes is dependent upon the presence of input RNA.

EXAMPLE 21 The Fate of the Target RNA in the Invader-Directed CleavageReaction

In this example, the fate of the RNA target in the invader-directedcleavage reaction was examined. As shown above in Example 1D, when RNAsare hybridized to DNA oligonucleotides, the 5′ nucleases associated withDNA polymerases can be used to cleave the RNAs; such cleavage can besuppressed when the 5′ arm is long or when it is highly structured[Lyamichev et al. (1993) Science 260:778 and U.S. Pat. No. 5,422,253,the disclosure of which is herein incorporated by reference]. In thisexperiment, the extent to which the RNA target would be cleaved by thecleavage agents when hybridized to the detection oligonucleotides (i.e.,the probe and invader oligonucleotides) was examined using reactionssimilar to those described in Example 20, performed usingfluorescein-labeled RNA as a target.

Transcription reactions were performed as described in Example 20 withthe exception that 2% of the UTP in the reaction was replaced withfluorescein-12-UTP (Boehringer Mannheim) and 5.3 μg of the amplicon wasused in a 100 μl reaction. The transcription reaction was incubated for2.5 hours, after which the DNA template was destroyed by the addition of5-6 μl of RQ1 RNAse-free DNAse (1 unit/μl) according to the RiboMAX™ kitinstructions. The organic extraction was omitted and the RNA wascollected by the addition of 10 μl of 3M NaOAc, pH 5.2 and 110 μl of100% isopropanol. Following a 5 min. incubation at 4° C., theprecipitate was collected by centrifugation, washed once with 80%ethanol and dried under vacuum. The resulting RNA was dissolved in 100μl of nuclease-free water. 50% of the sample was purified byelectrophoresis through a 8% denaturing polyacrylamide gel (19:1cross-linked), containing 7 M urea, in a buffer of 45 mM Tris-Borate, pH8.3, 1.4 mM EDTA. The gel slice containing the full-length material wasexcised and the RNA was eluted by soaking the slice overnight at 4° C.in 200 μl of 10 mM Tris-Cl, pH 8.0, 0.1 mM EDTA and 0.3 M NaOAc. The RNAwas then precipitated by the addition of 2.5 volumes of 100% ethanol.After incubation at −20° C. for 30 min., the precipitates were recoveredby centrifugation, washed once with 80% ethanol and dried under vacuum.The RNA was dissolved in 25 μl of nuclease-free water and thenquantitated by UV absorbance at 260 nm.

Samples of the purified RNA target were incubated for 5 or 30 min. inreactions that duplicated the Cleavase® A/G and DNAPTth invaderreactions described in Example 20 with the exception that the reactionslacked probe and invader oligonucleotides. Subsequent analysis of theproducts showed that the RNA was very stable, with a very slightbackground of non-specific degradation, appearing as a gray backgroundin the gel lane. The background was not dependent on the presence ofenzyme in the reaction.

Invader detection reactions using the purified RNA target were performedusing the probe/invader pair described in Example 20 (SEQ ID NOS:57 and58). Each reaction included 500 fmole of the target RNA, 5 pmoles of thefluorescein-labeled probe and 10 pmoles of the invader oligonucleotidein a buffer of 10 mM MOPS, pH 7.5 with 150 mM LiCl, 4 mM MnCl₂, 0.05%each Tween-20 and Nonidet-P40 and 39 units RNAsin® (Promega). Thesecomponents were combined and warmed to 50° C. and the reactions werestarted by the addition of either 53 ng of Cleavase® A/G or 5 polymeraseunits of DNAPTth. The fmal reaction volume was 10 μl. After 5 min at 50°C., 5 μl aliquots of each reaction were removed to tubes containing 4 μlof 95% formamide, 10 mM EDTA and 0.02% methyl violet. The remainingaliquot received a drop of ChillOut® evaporation barrier and wasincubated for an additional 25 min. These reactions were then stopped bythe addition of 4 μl of the above formamide solution. The products ofthese reactions were resolved by electrophoresis through separate 20%denaturing polyacrylamide gels (19:1 cross-linked), containing 7 M urea,in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. Followingelectrophoresis, the labeled reaction products were visualized using theFMBIO-100 Image Analyzer (Hitachi), with the resulting imager scansshown in FIGS. 53A (5 min reactions) and 53B (30 min. reactions).

In FIG. 53 the target RNA is seen very near the top of each lane, whilethe labeled probe and its cleavage products are seen just below themiddle of each panel. The FMBIO-100 Image Analyzer was used toquantitate the fluorescence signal in the probe bands. In each panel,lane 1 contains products from reactions performed in the absence of acleavage agent, lane 2 contains products from reactions performed usingCleavase® A/G and lane 3 contains products from reactions performedusing DNAPTth.

Quantitation of the fluorescence signal in the probe bands revealed thatafter a 5 min. incubation, 12% or 300 fmole of the probe was cleaved bythe Cleavase® A/G and 29% or 700 fmole was cleaved by the DNAPTth. Aftera 30 min. incubation, Cleavase® A/G had cleaved 32% of the probemolecules and DNAPTth had cleaved 70% of the probe molecules. (Theimages shown in FIGS. 53A and 53B were printed with the intensityadjusted to show the small amount of background from the RNAdegradation, so the bands containing strong signals are saturated andtherefore these images do not accurately reflect the differences inmeasured fluorescence)

The data shown in FIG. 53 clearly shows that, under invasive cleavageconditions, RNA molecules are sufficiently stable to be detected as atarget and that each RNA molecule can support many rounds of probecleavage.

EXAMPLE 22 Titration of Target RNA in the Invader-Directed CleavageAssay

One of the primary benefits of the invader-directed cleavage assay as ameans for detection of the presence of specific target nucleic acids isthe correlation between the amount of cleavage product generated in aset amount of time and the quantity of the nucleic acid of interestpresent in the reaction. The benefits of quantitative detection of RNAsequences was discussed in Example 20. In this example, we demonstratethe quantitative nature of the detection assay through the use ofvarious amounts of target starting material. In addition todemonstrating the correlation between the amounts of input target andoutput cleavage product, these data graphically show the degree to whichthe RNA target can be recycled in this assay

The RNA target used in these reactions was the fluorescein-labeledmaterial described in Example 21 (i.e., SEQ ID NO:56). Because theefficiency of incorporation of the fluorescein-12-UTP by the T7 RNApolymerase was not known, the concentration of the RNA was determined bymeasurement of absorbance at 260 nm, not by fluorescence intensity. Eachreaction comprised 5 pmoles of the fluorescein-labeled probe (SEQ IDNO:57) and 10 pmoles of the invader oligonudeotide (SEQ ID NO:58) in abuffer of 10 mM MOPS, pH 7.5 with 150 mM LiCl, 4 mM MnCl₂, 0.05% eachTween-20 and Nonidet-P40 and 39 units of RNAsin® (Promega). The amountof target RNA was varied from 1 to 100 pmoles, as indicated below. Thesecomponents were combined, overlaid with ChillOut® evaporation barrier(MJ Research) and warmed to 50° C.; the reactions were started by theaddition of either 53 ng of Cleavase® A/G or 5 polymerase units ofDNAPTth, to a final reaction volume of 10 μl. After 30 minutes at 50°C., reactions were stopped by the addition of 8 μl of 95% formamide, 10mM EDTA and 0.02% methyl violet. The unreacted markers in lanes 1 and 2were diluted in the same total volume (18 μl). The samples were heatedto 90° C. for 1 minute and 2.5 μl of each of these reactions wereresolved by electrophoresis through a 20% denaturing polyacrylamide gel(19:1 cross link) with 7M urea in a buffer of 45 mM Tris-Borate, pH 8.3,1.4 mM EDTA, and the labeled reaction products were visualized using theFMBIO-100 Image Analyzer (Hitachi), with the resulting imager scansshown in FIG. 54.

In FIG. 54, lanes 1 and 2 show 5 pmoles of uncut probe and 500 fmoles ofuntreated RNA, respectively. The probe is the very dark signal near themiddle of the panel, while the RNA is the thin line near the top of thepanel. These RNAs were transcribed with a 2% substitution offluorescein-12-UTP for natural UTP in the transcription reaction. Theresulting transcript contains 74 U residues, which would give an averageof 1.5 fluorescein lables per molecule. With one tenth the molar amountof RNA loaded in lane 2, the signal in lane 2 should be approximatelyone seventh (0.15×) the fluorescence intensity of the probe in lane 1.Measurements indicated that the intensity was closer to one fortieth,indicating an efficiency of label incorporation of approximately 17%.Because the RNA concentration was verified by A260 measurement this doesnot alter the experimental observations below, but it should be notedthat the signal from the RNA and the probes does not accurately reflectthe relative amounts in the reactions.

The reactions analyzed in lanes 3 through 7 contained 1, 5, 10, 50 and100 fmoles of target, respectively, with cleavage of the probeaccomplished by Cleavase® A/G. The reactions analyzed in lanes 8 through12 repeated the same array of target amounts, with cleavage of the probeaccomplished by DNAPTth. The boxes seen surrounding the product bandsshow the area of the scan in which the fluorescence was measured foreach reaction. The number of fluorescence units detected with each boxis indicated below each box; background florescence was also measured.

It can be seen by comparing the detected fluorescence in each lane thatthe amount of product formed in these 30 minute reactions can becorrelated to the amount of target material. The accumulation of productunder these conditions is slightly enhanced when DNAPTth is used as thecleavage agent, but the correlation with the amount of target presentremains. This demonstrates that the invader assay can be used as a meansof measuring the amount of target RNA within a sample.

Comparison of the fluorescence intensity of the input RNA with that ofthe cleaved product shows that the invader-directed cleavage assaycreates signal in excess of the amount of target, so that the signalvisible as cleaved probe is far more intense than the representing thetarget RNA. This further confirms the results described in Example >>,in which it was demonstrated that each RNA molecule could be used manytimes.

EXAMPLE 23 Detection of DNA by Charge Reversal

The detection of specific targets is achieved in the invader-directedcleavage assay by the cleavage of the probe oligonucleotide. In additionto the methods described in the preceding examples, the cleaved probemay be separated from the uncleaved probe using the charge reversaltechnique described below. This novel separation technique is related tothe observation that positively charged adducts can affect theelectrophoretic behavior of small oligonucleotides because the charge ofthe adduct is significant relative to charge of the whole complex.Observations of abberant mobility due to charged adducts have beenreported in the literature, but in all cases found, the applicationspursued by other scientists have involved making oligonucleotides largerthan enzymatic extension. As the negatively charged nucleotides areadded on, the positive influence of the adduct is reduced toinsignificance. As a result, the effects of positively charged adductshave been dismissed and have received infinitesimal notice in theexisting literature.

This observed effect is of particular utility in assays based on thecleavage of DNA molecules. When an oligonucleotide is shortened throughthe action of a Cleavase® enzyme or other cleavage agent, the positivecharge can be made to not only significantly reduce the net negativecharge, but to actually override it, effectively “flipping” the netcharge of the labeled entity. This reversal of charge allows theproducts of target-specific cleavage to be partitioned from uncleavedprobe by extremely simple means. For example, the products of cleavagecan be made to migrate towards a negative electrode placed at any pointin a reaction vessel, for focused detection without gel-basedelectrophoresis. When a slab gel is used, sample wells can be positionedin the center of the gel, so that the cleaved and uncleaved probes canbe observed to migrate in opposite directions. Alternatively, atraditional vertical gel can be used, but with the electrodes reversedrelative to usual DNA gels (i.e., the positive electrode at the top andthe negative electrode at the bottom) so that the cleaved moleculesenter the gel, while the uncleaved disperse into the upper reservoir ofelectrophoresis buffer.

An additional benefit of this type of readout is that the absolutenature of the partition of products from substrates means that anabundance of uncleaved probe can be supplied to drive the hybridizationstep of the probe-based assay, yet the unconsumed probe can besubtracted from the result to reduce background.

Through the use of multiple positively charged adducts, syntheticmolecules can be constructed with sufficient modification that thenormally negatively charged strand is made nearly neutral. When soconstructed, the presence or absence of a single phosphate group canmean the difference between a net negative or a net positive charge.This observation has particular utility when one objective is todiscriminate between enzymatically generated fragments of DNA, whichlack a 3 phosphate, and the products of thermal degradation, whichretain a 3 phosphate (and thus two additional negative charges).

-   -   a) Characterization of the Products of Thermal Breakage of DNA        Oligonucleotides

Thermal degradation of DNA probes result in high background which canobscure signals generated by specific enzymatic cleavage, decreasing thesignal-to-noise ratio. To better understand the nature of DNA thermaldegradation products, we incubated the 5′ tetrachloro-fluorescein(TET)-labeled oligonucleotides 78 (SEQ ID NO:59) and 79 (SEQ ID NO:60)(100 pmole each) in 50 μl 10 mM NaCO₃ (pH 10.6), 50 mM NaCl at 90° C.for 4 hours. To prevent evaporation of the samples, the reaction mixturewas overlaid with 50 μl of ChillOut® 14 liquid wax (MJ Research). Thereactions were then divided in two equal aliquots (A and B). Aliquot Awas mixed with 25 μl of methyl violet loading buffer and Aliquot B wasdephosphorylated by addition of 2.5 μl of 100 mM MgCl₂ and 1 μl of 1unit/μl Calf Intestinal Alkaline Phosphatase (CIAP) (Promega), withincubation at 37° C. for 30 min. after which 25 μl of methyl violetloading buffer was added. One microliter of each sample was resolved byelectrophoresis through a 12% polyacrylamide denaturing gel and imagedas described in Example 21; a 585 nm filter was used with the FMBIOImage Analyzer. The resulting imager scan is shown in FIG. 55. In FIG.55, lanes 1-3 contain the TET-labeled oligonucleotide 78 and lanes 4-6contain the TET-labeled oligonucleotides 79. Lanes 1 and 4 containproducts of reactions which were not heat treated. Lanes 2 and 5 containproducts from reactions which were heat treated and lanes 3 and 6contain products from reactions which were heat treated and subjected tophosphatase treatment.

As shown in FIG. 55, heat treatment causes significant breakdown of the5′-TET-labeled DNA, generating a ladder of degradation (FIG. 55, lanes2, 3, 5 and 6). Band intensities correlate with purine and pyrimidinebase positioning in he oligonucleotide sequences, indicating thatbackbone hydrolysis may occur through formation of a basic intermediateproducts that faster rates for purines then for pyrimidines [Lindahl andKarlström (1973) Biochem. 12:151].

Dephosphorylation decreases the mobility of all products generated bythe thermal degradation process, with the most pronounced effectobserved for the shorter products (FIG. 55, lanes 3 and 6). Thisdemonstrates that thermally degraded products possess a 3′ end terminalphosphoryl group which can be removed by dephosphorylation with CIAP.Removal of the phosphoryl group decreases the overall negative charge by2. Therefore, shorter products which have a small number of negativecharges are influenced to a greater degree upon the removal of twocharges. This leads to a larger mobility shift in the shorter productsthan that observed for the larger species.

The fact that the majority of thermally degraded DNA products contain 3′end phosphate groups and Cleavase® enzyme-generated products do notallowed the development of simple isolation methods for productsgenerated in the invader-directed cleavage assay. The extra two chargesfound in thermal breakdown products do not exist in the specificcleavage products. Therefore, if one designs assays that producespecific products which contain a net positive charge of one or two,then similar thermal breakdown products will either be negative orneutral. The difference can be used to isolate specific products byreverse charge methods as shown below.

-   -   b) Dephosphorylation of Short Amino-Modified Oligonucleotides        can Reverse the Net Charge of the Labeled Product

To demonstrate how oligonucleotides can be transformed from net negativeto net positively charged compounds, the four short amino-modifiedoligonucleotides labeled 70, 74, 75 and 76 and shown in FIGS. 56-58 weresynthesized (FIG. 56 shows both oligonucleotides 70 and 74). All fourmodified oligonucleotides possess Cy-3 dyes positioned at the 5′-endwhich individually are positively charged under reaction and isolationconditions described in this example. Compounds 70 and 74 contain twoamino modified thymidines that, under reaction conditions, displaypositively charged R-NH₃ ⁺ groups attached at the C5 position through aC₁₀ or C₆ linker, respectively. Because compounds 70 and 74 are 3′-endphosphorylated, they consist of four negative charges and three positivecharges. Compound 75 differs from 74 in that the internal C₆ aminomodified thymidine phosphate in 74 is replaced by a thymidine methylphosphonate. The phosphonate backbone is uncharged and so there are atotal of three negative charges on compound 75. This gives compound 75 anet negative one charge. Compound 76 differs from 70 in that theinternal amino modified thymidine is replaced by an internal cytosinephosphonate. The pK_(a) of the N3 nitrogen of cytosine can be from 4 and7. Thus, the net charges of this compound, can be from −1 to 0 dependingon the pH of the solution. For the simplicity of analysis, each group isassigned a whole number of charges, although it is realized that,depending on the pK_(a) of each chemical group and ambient pH, a realcharge may differ from the whole number assigned. It is assumed thatthis difference is not significant over the range of pHs used in theenzymatic reactions studied here.

Dephosphorylation of these compounds, or the removal of the 3′ endterminal phosphoryl group, results in elimination of two negativecharges and generates products that have a net positive charge of one.In this experiment, the method of isoelectric focusing (IEF) was used todemonstrate a change from one negative to one positive net charge forthe described substrates during dephosphorylation.

Substrates 70, 74, 75 and 76 were synthesized by standardphosphoramidite chemistries and deprotected for 24 hours at 22° C. in 14M aqueous ammonium hydroxide solution, after which the solvent wasremoved in vacuo. The dried powders were resuspended in 200 μl of H₂Oand filtered through 0.2 μm filters. The concentration of the stocksolutions was estimated by UV-absorbance at 261 nm of samples diluted200-fold in H₂O using a spectrophotometer (Spectronic Genesys 2, MiltonRoy, Rochester, N.Y.).

Dephosphorylation of compounds 70 and 74, 75 and 76 was accomplished bytreating 10 μl of the crude stock solutions (ranging in concentrationfrom approximately 0.5 to 2 mM) with 2 units of CIAP in 100 μl of CIAPbuffer (Promega) at 37° C. for 1 hour. The reactions were then heated to75° C. for 15 min. in order to inactivate the CIAP. For clarity,dephosphorylated compounds are designated ‘dp’. For example, afterdephosphorylation, substrate 70 becomes 70dp.

To prepare samples for IEF experiments, the concentration of the stocksolutions of substrate an dephosphorylated product were adjusted to auniform absorbance of 8.5×10⁻³ at 532 nm by dilution with water. Twomicrofilters of each sample were analyzed by IEF using a PhastSystemelectrophoresis unit (Pharmacia) and PhastGel IEF 3-9 media (Pharmacia)according to the manufacturer's protocol. Separation was performed at15° C. with the following program: pre-run; 2,000 V, 2.5 mA, 3.5 W, 75Vh; load; 200 V, 2.5 mA, 3.5 W, 15 Vh; run; 2,000 V; 2.5 mA; 3.5 W, 130Vh. After separation, samples were visualized by using the FMBIO ImageAnaylzer (Hitachi) fitted with a 585 nm filter. The resulting imagerscan is shown in FIG. 59.

FIG. 59 shows results of IEF separation of substrates 70, 74, 75 and 76and their dephosphorylated products. The arrow labeled “Sample LoadingPosition” indicates a loading line, the ‘+’ sign shows the position ofthe positive electrode and the ‘−’ sign indicates the position of thenegative electrode.

The results shown in FIG. 59 demonstrate that substrates 70, 74, 75 and76 migrated toward the positive electrode, while the dephosphorylatedproducts 70dp, 74dp, 75dp and 76dp migrated toward negative electrode.The observed differences in mobility direction was in accord withpredicted net charge of the substrates (minus one) and the products(plus one). Small perturbations in the mobilities of the phosphorylatedcompounds indicate that the overall pIs vary. This was also true for thedephosphorylated compounds. The presence of the cytosine in 76dp, forinstance, moved this compound further toward the negative electrodewhich was indicative of a higher overall pI relative to the otherdephosphorylated compounds. It is important to note that additionalpositive charges can be obtained by using a combination of natural aminomodified bases (70dp and 74dp) along with uncharged methylphosphonatebridges (products 75dp and 76dp).

The results shown above demonstrate that the removal of a singlephosphate group can flip that net charge of an oligonucleotide to causereversal in an electric field, allowing easy separation of products, andthat the precise base composition of the oligonucleotides affectabsolute mobility but not the charge-flipping effect.

EXAMPLE 23 Detection of Specific Cleavage Products in theInvader-Directed Cleavage Reaction by Charge Reversal

In this example the ability to isolate products generated in theinvader-directed cleavage assay from all other nucleic acids present inthe reaction cocktail was demonstrated using charge reversal. Thisexperiment utilized the following Cy3-labeled oligonucleotide:5′-Cy3-AminoT-CTTTTCACCAGCGAGACGGG-3′ (SEQ ID NO:61; termed “oligo 61”).Oligo 61 was designed to release upon cleavage a net positively chargedlabeled product. To test whether or not a net positively charged 5′-endlabeled product would be recognized by the Cleavase® enzymes in theinvader-directed cleavage assay format, probe oligo 61 (SEQ ID NO:61)and invading oligonucleotide 67 (SEQ ID NO:62) were chemicallysynthesized on a DNA synthesizer (ABI 391) using standardphosphoramidite chemistries and reagents obtained from Glen Research(Sterling, Va.).

Each assay reaction compound 100 fmoles of M13mp18 single stranded DNA,10 pmoles each of the probe (SEQ ID NO:61) and invader (SEQ ID NO:62)oligonucleotides, and 20 units of Cleavase® A/G in a 10 μl solution of10 mM MOPS, pH 7.4 with 100 mM KCl. Samples were overlaid with mineraloil to prevent evaporation. The samples were brought to either 50° C.,55° C., 60° C., or 65° C. and cleavage was initiated by the addition of1 μl of 40 mM MnCl₂. Reactions were allowed to proceed for 25 minutesand then were terminated by the addition of 10 μl of 95% formamidecontaining 20 mM EDTA and 0.02% methyl violet. The negative controlexperiment lacked the target M13mp18 and was run at 60° C. Fivemicroliters of each reaction were loaded into separate wells of a 20%denaturing polyacrylamide gel (cross-lined 29:1) with 8 M urea in abuffer containing 45 mM Tris-Borate (pH 8.3) and 1.4 mM EDTA. Anelectric field of 20 watts was applied for 30 minutes, with theelectrodes oriented as indicated in FIG. 60B (i.e., in reverseorientation). The products of these reactions were visualized using theFMBIO fluorescence imager and the resulting imager scan is shown in FIG.60B.

FIG. 60A provides a schematic illustration showing an alignment of theinvader (SEQ ID NO:61) and probe (SEQ ID NO:62) along the target M13mp18DNA; only 53 bases of the M13mp18 sequence is shown (SEQ ID NO:63). Thesequence of the invader oligonucleotide is displayed under the M13mp18target and an arrow is used above the M13mp18 sequence to indicate theposition of the invader relative to the probe and target. As shown inFIG. 60A, the invader and probe oligonucleotides share a 2 base regionof overlap.

In FIG. 60B, lanes 1-6 contain reactions performed at 50° C., 55° C.,60° C., and 65° C., respectively; lane 5 contained the control reaction(lacking target). In FIG. 60B, the products of cleavage are seen as darkbands in the upper half of the panel; the faint lower band seen appearsin proportion to the amount of primary product produced and, while notlimiting the invetion to a particular mechanism, may represent cleavageone nucleotide into the duplex. The uncleaved probe does not enter thegel and is thus not visible. The control lane showed no detectablesignal over background (lane 5). As expected in an invasive cleavagereaction, the rate of accumulation of specific cleavage product wastemperature-dependent. Using these particular oligonucleotides andtarget, the fastest rate of accumulation of product was observed at 55°C. (lane 2) and very little product observed at 65° C. (lane 4).

When incubated for extended periods at high temperature, DNA probes canbreak non-specifically (i.e., suffer thermal degradation) and theresulting fragments contribute an interfering background to theanalysis. The products of such thermal breakdown are distributed fromsingle-nucleotides up to the full length probe. In this experiment, theability of charge based separation of cleavage products (i.e., chargereversal) would allow the sensitive separation of the specific productsof target-dependent cleavage from probe fragments generated by thermaldegradation was examined.

To test the sensitivity limit of this detection method, the targetM13mp18 DNA was serially diluted ten fold over than range of 1 fmole to1 amole. The invader and probe oligonucleotides were those describedabove (i.e., SEQ ID NOS:61 and 62). The invasive cleavage reactions wererun as described above the following modifications: the reactions wereperformed at 55° C., 250 mM or 100 mM KGlu was used in place of the 100mM KCl and only 1 pmole of the invader oligonucleotide was added. Thereactions were initiated as described above and allowed to progress for12.5 hours. A negative control reaction which lacked added M13m18 targetDNA was also run. The reactions were terminated by the addition of 10 μlof 95% formamide containing 20 mM EDTA and 0.02% methyl violet, and 5 μlof these mixtures were electrophoresed and visualized as describedabove. The resulting imager scan is shown in FIG. 61.

In FIG. 61, lane 1 contains the regative control; lanes 2-5 containreactions performed using 100 mM KGlu; lanes 6-9 contain reactionsperformed using 250 mM KGlu. The reactions resolved in lanes 2 and 6contained 1 fmole of target DNA; those in lanes 3 and 7 contained 100amole of target; those in lanes 4 and 8 contained 10 amole of target andthose in lanes 5 and 9 contained 1 amole of target. The results shown inFIG. 61 demonstrate that the detection limit using charge reversal todetect the production of specific cleavage products in an invasivecleavage reaction is at or below 1 attomole or approximately 6.02×10⁵target molecules. No detectable signal was observed in the control lane,which indicates that non-specific hydrolysis or other breakdown productsdo not migrate in the same direction as enzyme-specific cleavageproducts. The excitation and emission maxima for Cy3 are 554 and 568,respectively, while the FMBIO Imager Analyzer excites at 532 and detectsat 585. Therefore, the limit of detection of specific cleavage productscan be improved by the use of more closely matched excitation source anddetection filters.

EXAMPLE 24 Devices and Methods for the Separation and Detection ofCharged Reaction Products

This example is directed at methods and devices for isolating andconcentrating specific reaction products produced by enzymatic reactionsconducted in solution whereby the reactions generate charged productsfrom either a charge neutral substrate or a substrate bearing theopposite charge borne by the specific reaction product. The methods anddevices of this example allow isolation of, for example, the productsgenerated by the invader-directed cleavage assay of the presentinvention.

The methods and devices of this example are based on the principle thatwhen an electric field is applied to a solution of charged molecules,the migration of the molecules toward the electrode of the oppositecharge occurs very rapidly. If a matrix or other inhibitory material isintroduced between the charged molecules and the electrode of oppositecharge such that this rapid migration is dramatically slowed, the firstmolecules to reach the matrix will be nearly stopped, thus allowing thelagging molecules to catch up. In this way a dispersed population ofcharged molecules in solution can be effectively concentrated into asmaller volume. By tagging the molecules with a detectable moiety (e.g.,a fluorescent dye), detection is facilitated by both the concentrationand the localization of the analytes. This example illustrates twoembodiments of devices contemplated by the present invention; of course,variations of these devices will be apparent to those skilled in the artand are within the spirit and scope of the present invention.

FIG. 62 depicts one embodiment of a device for concentrating thepositively-charged products generated using the methods of the presentinvention. As shown in FIG. 62, the device comprises a reaction tube(10) which contains the reaction solution (11). One end of each of twothin capillaries (or other tubes with a hollow core) (13A and 13B) aresubmerged in the reaction solution (11). The capillaries (13A and 13B)may be suspended in the reaction solution (11) such that they are not incontact with the reaction tube itself; one appropriate method ofsuspending the capillaries is to hold them in place with clamps (notshown). Alternatively, the capillaries may be suspended in the reactionsolution (11) such that they are in contact with the reaction tubeitself. Suitable capillaries include glass capillary tubes commonlyavailable from scientific supply companies (e.g., Fisher Scientific orVWR Scientific) or from medical supply houses that carry materials forblood drawing and analysis. Though the present invention is not limitedto capillaries of any particular inner diameter, tubes with innerdiameters of up to about ⅛ inch (approximately 3 mm) are particularlypreferred for use with the present invention; for example Kimble No.73811-99 tubes (VWR Scientific) have an inner diameter of 1.1 mm and area suitable type of capillary tube. Although the capillaries of thedevice are commonly composed of glass, any nonconductive tubularmaterial, either rigid or flexible, that can contain either a conductivematerial or a trapping material is suitable for use in the presentinvention. One example of a suitable flexible tube is Tygon® clearplastic tubing (Part No. R3603; inner diameter= 1/16 inch; outerdiameter= 1/8 inch).

As illustrated in FIG. 62, capillary 13A is connected to the positiveelectrode of a power supply (20) (e.g., a controllable power supplyavailable through the laboratory suppliers listed above or throughelectronics supply houses like Radio Shack) and capillary 13B isconnected to the negative electrode of the power supply (20). Capillary13B is filled with a trapping material (14) capable of trapping thepositively-charged reaction products by allowing minimal migration ofproducts that have entered the trapping material (14). Suitable trappingmaterials include, but are not limited to, high percentage (e.g., about20%) acrylamide polymerized in a high salt buffer (0.5 M or highersodium acetate or similar salt); such a high percentage polyacrylamidematrix dramatically slows the migration of the positively-chargedreaction products. Alternatively, the trapping material may comprise asolid, negatively-charged matrix, such as negatively-charged latexbeads, that can bind the incoming positively-charged products. It shouldbe noted that any amount of trapping material (14) capable of inhibitingany concentrating the positively-charged reaction products may be used.Thus, while the capillary 13B in FIG. 62 only contains trapping materialin the lower, submerged portion of the tube, the trapping material (14)can be present in the entire capillary (13B); similarly, less trappingmaterial (14) could be present than that shown in FIG. 62 because thepositively-charged reaction products generally accumulate within a verysmall portion of the bottom of the capillary (13B). The amount oftrapping material need only be sufficient to make contact with thereaction solution (11) and have the capacity to collect the reactionproducts. When capillary 13B is not completely filled with the trappingmaterial, the remaining space is filled with any conductive material(15); suitable conductive materials are discussed below.

By comparison, the capillary (13A) connected to the positive electrodeof the power supply 20 may be filled with any conductive material (15;indicating by the hatched lines in FIG. 62). This may be the samplereaction buffer (e.g., 10 mM MOPS, pH 7.5 with 150 mM LiCl, 4 mM MnCl₂),a standard electrophoresis buffer (e.g., 45 mM Tris-Borate, pH 8.3, 1.4mM EDTA), or the reaction solution (11) itself. The conductive material(15) is frequently a liquid, but a semi-solid material (e.g., a gel) orother suitable material might be easier to use and is within the scopeof the present invention. Moreover, that trapping material used in theother capillary (i.e., capillary 13B) may also be used as the conductivematerial. Conversely, it should be noted that the same conductivematerial used in the capillary (13A) attached to the positive electrodemay also be used in capillary 13B to fill the space above the regioncontaining the trapping material (14) (see FIG. 62).

The top end of the capillaries (13A and 13B) is connected to theappropriate electrode of the power supply (20) by electrode wire (18) orother suitable material. Fine platinum wire (e.g., 0.1 to 0.4 mm, AesarJohnson Matthey, Ward Hill, Mass.) is commonly used as conductive wirebecause it does not corrode under electrophoresis conditions. Theelectrode wire (18) can be attached to the capillaries (13A and 13B) bya nonconductive adhesive (not shown), such as the silicone adhesivesthat are commonly sold in hardware stores for sealing plumbing fixtures.If the capillaries are constructed of a flexible material, the electrodewire (18) can be secured with a small hose clamp or constricting wire(not shown) to compress the opening of the capillaries around theelectrode wire. If the conducting material (15) is a gel, an electrodewire (18) can be embedded directly in the gel within the capillary.

The cleavage reaction is assembled in the reaction tube (10) and allowedto proceed therein as described in proceeding examples (e.g., Examples22-23). Though not limited to any particular volume of reaction solution(11), a preferred volume is less than 10 ml and more preferably lessthan 0.1 ml. The volume need only be sufficient to permit contact withboth capillaries. After the cleavage reaction is completed, an electricfield is applied to the capillaries by turning on the power source (20).As a result, the positively-charged products generated in the course ofthe invader-directed cleavage reaction which employs an oligonucleotide,which when cleaved, generates a positively charged fragment (describedin Ex. 23) but when uncleaved bears a net negative charge, migrate tothe negative capillary, where their migration is slowed or stopped bythe trapping material (14), and the negatively-charged uncut andthermally degraded probe molecules migrate toward the positiveelectrode. Through the use of this or a similar device, thepositively-charged products of the invasive cleavage reaction areseparated from the other material (i.e., uncut and thermally degradedprobe) and concentrated from a large volume. Concentration of theproduct in a small amount of trapping material (14) allows forsimplicity of detection, with a much higher signal-to-noise ratio thanpossible with detection in the original reaction volume. Because theconcentrated product is labelled with a detectable moiety like afluorescent dye, a commercially-available fluorescent plate reader (notshown) can be used to ascertain the amount of product. Suitable platereaders include both top and bottom laser readers. Capillary 13B can bepositioned with the reaction tube (10) at any desired position so as toaccommodate use with either a top or a bottom plate reading device.

In the alternative embodiment of the present invention depicted in FIG.63, the procedure described above is accomplished by utilizing only asingle capillary (13B). The capillary (13B) contains the trappingmaterial (14) described above and is connected to an electrode wire(18), which in turn is attached to the negative electrode of a powersupply (20). The reaction tube (10) has an electrode (25) embedded intoits surface such that one surface of the electrode is exposed to theinterior of the reaction tube (10) and another surface is exposed to theexterior of the reaction tube. The surface of the electrode (25) on theexterior of the reaction tube is in contact with a conductive surface(26) connected to the positive electrode of the power supply (20)through an electrode wire (18). Variations of the arrangement depictedin FIG. 63 are also contemplated by the present invention. For example,the electrode (25) may be in contact with the reaction solution (11)through the use of a small hole in the reaction tube (10); furthermore,the electrode wire (18) can be directly attached to the electrode wire(18), thereby eliminating the conductive surface (26).

As indicated in FIG. 63, the electrode (25) is embedded in the bottom ofa reaction tube (10) such that one or more reaction tubes may be set onthe conductive surface (26). This conductive surface could serve as anegative electrode for multiple reaction tubes; such a surface withappropriate contacts could be applied through the use of metal foils(e.g., copper or platinum, Aesar Johnson Matthey, Ward Hill, Mass.) inmuch the same way contacts are applied to circuit boards. Because such asurface contact would not be exposed to the reaction sample directly,less expensive metals, such as the copper could be used to make theelectrical connections.

The above devices and methods are not limited to separation andconcentration of positively charged oligonucleotides. As will beapparent to those skilled in the art, negatively charged reactionproducts may be separated from neutral or positively charged reactantsusing the above device and methods with the exception that capillary 13Bis attached to the positive electrode of the power supply (20) andcapillary 13A or alternatively, electrode 25, is attached to thenegative electrode of the power supply (20).

EXAMPLE 25 Primer-Directed and Primer Independent Cleavage Occur at theSame Site When the Primer Extends to the 3′ Side of a Mismatched“Bubble” in the Downstream Duplex

As discussed above in Example 1, the presence of a primer upstream of abifurcated duplex can influence the site of cleavage, and the existenceof a gap between the 3′ end of the primer and the base of the duplex cancause a shift of the cleavage site up the unpaired 5′ arm of thestructure (see also Lyamichev et al., supra and U.S. Pat. No.5,422,253). The resulting non-invasive shift of the cleavage site inresponse to a primer is demonstrated in FIGS. 9, 10 and 11, in which theprimer used left a 4-nucleotide gap (relative to the base of theduplex). In FIGS. 9-11, all of the “primer-directed” cleavage reactionsyielded at 21 nucleotide product, while the primer-independent cleavagereactions yielded a 25 nucleotide product. The site of cleavage obtainedwhen the primer was extended to the base of the duplex, leaving no gapwas examined. The results are shown in FIG. 64 (FIG. 64 is areproduction of FIG. 2C in Lyamichev et al. These data were derived fromthe cleavage of the structure shown in FIG. 6, as described inExample 1. Unless otherwise specified, the cleavage reactions comprised0.01 pmoles of heat-denatured, end-labeled hairpin DNA (with theunlabeled complementary strand also present), 1 pmole primer[complementary to the 3′ arm shown in FIG. 6 and having the sequence:5′-GAAT TCGATTTAGGTGACACTATAGAATACA (SEQ ID NO:64)] and 0.5 units ofDNAPTaq (estimated to be 0.026 pmoles) in a total volume of 10 μl of 10mM Tris-Cl, pH 8.5, and 1.5 mM MgCl₂ and 50 mM KCl. The primer wasomitted from the reaction shown in the first lane of FIG. 64 andincluded in lane 2. These reactions were incubated at 55° C. for 10minutes. Reactions were initiated at the final reaction temperature bythe addition of either the MgCl₂ or enzyme. Reactions were stopped attheir incubation temperatures by the addition of 8 μl of 95% formamidewith 20 mM EDTA and 0.05% marker dyes.

FIG. 64 is an autoradiogram that indicates the effects on the site ofcleavage of a bifurcated duplex structure in the presence of a primerthat extends to the base of the hairpin duplex. The size of the releasedcleavage product is shown to the left (i.e., 25 nucleotides). Adideoxynucleotide sequencing ladder of the cleavage substrate is shownon the right as a marker (lanes 3-6).

These data show that the presence of a primer that is adjacent to adownstream duplex (lane 2) produces cleavage at the same site as seen inreactions performed in the absence of the primer (lane 1) (see FIGS. 9Aan B, 10B and 11A for additional comparisons). When the 3′ terminalnucleotides of the upstream oligonucleotide can base pair to thetemplate strand but are not homologous to the displaced strand in theregion immediately upstream of the cleavage site (i.e., when theupstream oligonucleotide is opening up a “bubble” in the duplex), thesite to which cleavage is apparently shifted is not wholly dependent onthe presence of an upstream oligonucleotide.

As discussed above in the Background section and in Table 1, therequirement that two independent sequences be recognized in an assayprovides a highly desirable level of specificity. In the invasivecleavage reactions of the present invention, the invader and probeoligonucleotides must hybridize to the target nucleic acid with thecorrect orientation and spacing to enable the production of the correctcleavage product. When the distinctive pattern of cleavage is notdependent on the successful alignment of both oligonucleotides in thedetection system these advantages of independent recognition are lost.

EXAMPLE 26 Invasive Cleavage and Primer-Directed Cleavage When there isOnly Partial Homology in the “X” Overlap Region

While not limiting the present invention to any particular mechanism,invasive cleavage occurs when the site of cleavage is shifted to a sitewithin the duplex formed between the probe and the target nucleic acidin a manner that is dependent on the presence of an upstreamoligonucleotide which shares a region of overlap with the downstreamprobe oligonucleotide. In some instances, the 5′ region of thedownstream oligonucleotide may not be completely complementary to thetarget nucleic acid. In these instances, cleavage of the probe may occurat an internal site within the probe even in the absence of an upstreamoligonucleotide (in contrast to the base-by-base nibbling seen when afully paired probe is used without an invader). Invasive cleavage ischaracterized by an apparent shifting of cleavage to a site within adownstream duplex that is dependent on the presence of the invaderoligonucleotide.

A comparison between invasive cleavage and primer-directed cleavagem maybe illustrated by comparing the expected cleavage sites of a set ofprobe oligonucleotides having decreasing degrees of complementarity tothe target strand in the 5′ region of the probe (i.e., the region thatoverlaps with the invader). A simple test, similar to that performed onthe hairpin substrate above (Ex. 25), can be performed to compareinvasive cleavage with the non-invasive primer-directed cleavagedescribed above. Such a set of test oligonucleotides is diagrammed inFIG. 65. The structures shown in FIG. 65 are grouped in pairs, labeled“a”, “b”, “c”, and “d”. Each pair has the same probe sequence annealedto the target strand (SEQ ID NO:65), but the top structure of each pairis drawn without an upstream oligonucleotide, while the bottom structureincludes this oligonucleotide (SEQ ID NO:66). The sequences of theprobes shown in FIGS. 64 a-64 d are listed in SEQ ID NOS:43, 67, 68 and69, respectively. Probable sites of cleavage are indicated by the blackarrowheads. (It is noted that the precise site of cleavage on each ofthese structures may vary depending on the choice of cleavage agent andother experimental variables. These particular sites are provided forillustrative purposes only).

To conduct this test, the site of cleavage of each probe is determinedboth in the presence and the absence of the upstream oligonucleotide, inreaction conditions such as those described in Example 19. The productsof each pair of reactions are then be compared to determine whether thefragment released from the 5′ end of the probe increases in size whenthe upstream oligonucleotide is included in the reaction.

The arrangement shown in FIG. 65 a, in which the probe molecule iscompletely complementary to the target strand, is similar to that shownin FIG. 32. Treatment of the top structure with the 5′ nuclease of a DNApolymerase would cause exonucleolytic nibbling of the probe (i.e., inthe absence of the upstream oligonucleotide). In contrast, inclusion ofan invader oligonucleotide would cause a distinctive cleavage shiftsimilar, to those observed in FIG. 33.

The arrangements shown in FIGS. 65 b and 65 c have some amount ofunpaired sequence at the 5′ terminus of the probe (3 and 5 bases,respectively). These small 5′ arms are suitable cleavage substrate forthe 5′ nucleases and would be cleaved within 2 nucleotide's of thejunction between the single stranded region and the duplex. In thesearrangements, the 3′ end of the upstream oligonucleotide shares identitywith a portion of the 5′ region of the probe which is complementary tothe target sequence (that is the 3′ end of the invader has to competefor binding to the target with a portion of the 5′ end of the probe).Therefore, when the upstream oligonucleotide is included it is thoughtto mediate a shift in the site of cleavage into the downstream duplex(although the present invention is not limited to any particularmechanism of action), and this would, therefore, constitute invasivecleavage. If the extreme 5′ nucleotides of the unpaired region of theprobe were able to hybridize to the target stand, the cleavage site inthe absence of the invader might change but the addition of the invaderoligonucleotide would still shift the cleavage site to the properposition.

Finally,in the arrangement shown in FIG. 65 d, the probe and upstreamoligonucleotides share no significant regions of homology, and thepresence of the upstream oligonucleotide would not compete for bindingto the target with the probe. Cleavage of the structures shown in FIG.64 d would occur at the same site with or without the upstreamoligonucleotide, and is thus would not constitute invasive cleavage.

By examining any upstream oligonucleotide/probe pair in this way, it caneasily be determined whether the resulting cleavage is invasive ormerely primer-directed. Such analysis is particularly useful when theprobe is not fully complementary to the target nucleic acid, so that theexpected result may not be obvious by simple inspection of thesequences.

From the above it is clear that the invention provides reagents andmethods to permit the detection and characterization of nucleic acidsequences and variations in nucleic acid sequences. The invader-directedcleavage reaction of the present invention provides an ideal directdetection method that combines the advantages of the direct detectionassays (e.g., easy quantification and minimal risk of carry-overcontamination) with the specificity provided by a dual oligonucleotidehybridization assay.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

1. A method for detecting the presence of a target nucleic acid moleculein a sample, comprising: a) incubating a sample with a thermostable 5′nuclease under conditions wherein a cleavage structure is formed, saidcleavage structure comprising: i) a synthetic target nucleic acid, saidsynthetic target nucleic acid comprising a first region and a secondregion, said second region downstream of and contiguous to said firstregion; ii) a first nucleic acid molecule, wherein at least a portion ofsaid first nucleic acid molecule is completely complementary to saidfirst region of said target nucleic acid; iii) a second nucleic acidmolecule comprising a 3′ portion and a 5′ portion, wherein said 5′portion is completely complementary to said second region of said targetnucleic acid; wherein said thermostable 5′ nuclease lacks synthesisactivity, and wherein at least a portion of said first nucleic acidmolecule is annealed to said first region of said synthetic targetnucleic acid, and wherein at least a portion of said second nucleic acidmolecule is annealed to said second region of said synthetic targetnucleic acid; b) cleaving said cleavage structure with said thermostable5′ nuclease so as to generate non-target cleavage product; and c)detecting the cleavage of said cleavage structure.
 2. The method ofclaim 1, wherein said detecting the cleavage of said cleavage structurecomprises detecting said non-target cleavage product.
 3. The method ofclaim 1, wherein said synthetic target nucleic acid comprises anamplified nucleic acid.
 4. The method of claim 3, wherein said amplifiednucleic acid is produced using a polymerase chain reaction.
 5. Themethod of claim 1, wherein said detecting the cleavage of said cleavagestructure comprises detection of fluorescence.
 6. The method of claim 1,wherein said detecting the cleavage of said cleavage structure comprisesdetection of mass.
 7. The method of claim 1, wherein said detecting thecleavage of said cleavage structure comprises detection of fluorescenceenergy transfer.
 8. The method of claim 1, wherein said detecting thecleavage of said cleavage structure comprises detection selected fromthe group consisting of detection of radioactivity, luminescence,phosphorescence, fluorescence polarization, and charge.
 9. The method ofclaim 1, wherein said first nucleic acid molecule is attached to a solidsupport.
 10. The method of claim 1, wherein said second nucleic acidmolecule is attached to a solid support.
 11. The method of claim 1,wherein said synthetic target nucleic acid comprises DNA.
 12. The methodof claim 1, wherein said cleaving step is conducted under isothermalconditions.
 13. The method of claim 1, wherein said thermostable 5′nuclease comprises a 5′ nuclease of a DNA polymerase.
 14. The method ofclaim 13, wherein said DNA polymerase is Taq DNA polymerase.
 15. Themethod of claim 1, wherein said 3′ portion of said second nucleic acidmolecule comprises an aromatic ring.
 16. The method of claim 1, whereinsaid 3′ portion of said second nucleic acid molecule comprises a 3′terminal nucleotide not complementary to said target nucleic acid. 17.The method of claim 1, wherein said 3′ portion of said second nucleicacid molecule consists of a single nucleotide.
 18. The method of claim17, wherein said single nucleotide is not complementary to said targetnucleic acid.
 19. The method of claim 17, wherein said single nucleotideis complementary to said target nucleic acid.
 20. The method of claim 6,wherein said 3′ terminal nucleotide comprises a naturally occurringnucleotide.
 21. The method of claim 16, wherein said 3′ terminalnucleotide comprises a nucleotide analog.
 22. The method of claim 1,wherein a plurality of said first nucleic acid molecule is provided,such that said first nucleic acid molecule is in concentration excesscompared to said target nucleic acid.
 23. The method of claim 1, whereina plurality of said second nucleic acid molecule is provided, such thatsaid second nucleic acid molecule is in concentration excess compared tosaid target nucleic acid.
 24. The method of claim 1, wherein said targetnucleic acid and said second nucleic acid molecule form a duplex, andwherein a plurality of said first nucleic acid molecule is provided suchthat said first nucleic acid molecule is in concentration excesscompared to said duplex.
 25. The method of claim 24, wherein saidcleaving said cleavage structure comprises cleaving said first nucleicacid molecule to generate non-target cleavage product.
 26. The method ofclaim 25, wherein said non-target cleavage product from said firstnucleic acid molecule is generated in concentration excess compared tosaid duplex.
 27. The method of claim 1, further comprising providing athird nucleic acid molecule complementary to a third portion of saidtarget nucleic acid upstream of said first portion of said first targetnucleic acid, wherein said cleavage structure comprises said thirdnucleic acid molecule.
 28. The method of claim 1, wherein saidthermostable 5′ nuclease is a FEN-1 nuclease.
 29. The method of claim 1,wherein said 3′ portion of said second nucleic acid molecule iscompletely complementary to said target nucleic acid.
 30. The method ofclaim 29, wherein during said incubating, said 3′ portion of said secondnucleic acid molecule partially displaces said portion of said firstnucleic acid molecule that is completely complementary to said firstregion of said target nucleic acid.
 31. A method for detecting thepresence of a target nucleic acid molecule in a sample, comprising: a)incubating a sample with a thermostable FEN-1 nuclease under conditionswherein a cleavage structure is formed, said cleavage structurecomprising: i) a synthetic target nucleic acid, said synthetic targetnucleic acid comprising a first region and a second region, said secondregion downstream of and contiguous to said first region; ii) a firstnucleic acid molecule, wherein at least a portion of said first nucleicacid molecule is completely complementary to said first region of saidtarget nucleic acid; iii) a second nucleic acid molecule comprising a 3′portion and a 5′ portion, wherein said 5′ portion is completelycomplementary to said second region of said target nucleic acid; whereinat least a portion of said first nucleic acid molecule is annealed tosaid first region of said synthetic target nucleic acid, and wherein atleast a portion of said second nucleic acid molecule is annealed to saidsecond region of said synthetic target nucleic acid; b) cleaving saidcleavage structure with said thermostable FEN-1 nuclease so as togenerate non-target cleavage product; and c) detecting the cleavage ofsaid cleavage structure.
 32. The method of claim 31, wherein saiddetecting the cleavage of said cleavage structure comprises detectingsaid non-target cleavage product.
 33. The method of claim 31, whereinsaid synthetic target nucleic acid comprises an amplified nucleic acid.34. The method of claim 33, wherein said amplified nucleic acid isproduced using a polymerase chain reaction.
 35. The method of claim 31,wherein said detecting the cleavage of said cleavage structure comprisesdetection of fluorescence.
 36. The method of claim 31, wherein saiddetecting the cleavage of said cleavage structure comprises detection ofmass.
 37. The method of claim 31, wherein said detecting the cleavage ofsaid cleavage structure comprises detection of fluorescence energytransfer.
 38. The method of claim 31, wherein said detecting thecleavage of said cleavage structure comprises detection selected fromthe group consisting of detection of radioactivity, luminescence,phosphorescence, fluorescence polarization, and charge.
 39. The methodof claim 31, wherein said first nucleic acid molecule is attached to asolid support.
 40. The method of claim 31, wherein said second nucleicacid molecule is attached to a solid support.
 41. The method of claim31, wherein said synthetic target nucleic acid comprises DNA.
 42. Themethod of claim 31, wherein said cleaving step is conducted underisothermal conditions.
 43. The method of claim 31, wherein said 3′portion of said second nucleic acid molecule comprises an aromatic ring.44. The method of claim 31, wherein said 3′ portion of said secondnucleic acid molecule comprises a 3′ terminal nucleotide notcomplementary to said target nucleic acid.
 45. The method of claim 31,wherein said 3′ portion of said second nucleic acid molecule consists ofa single nucleotide.
 46. The method of claim 45, wherein said singlenucleotide is not complementary to said target nucleic acid.
 47. Themethod of claim 45, wherein said single nucleotide is complementary tosaid target nucleic acid.
 48. The method of claim 44, wherein said 3′terminal nucleotide comprises a naturally occurring nucleotide.
 49. Themethod of claim 44, wherein said 3′ terminal nucleotide comprises anucleotide analog.
 50. The method of claim 31, wherein said 3′ portionof said second nucleic acid molecule is completely complementary to saidtarget nucleic acid.
 51. The method of claim 50, wherein during saidincubating, said 3′ portion of said second nucleic acid moleculepartially displaces said portion of said first nucleic acid moleculethat is completely complementary to said first region of said targetnucleic acid.
 52. The method of claim 31, wherein a plurality of saidfirst nucleic acid molecule is provided, such that said first nucleicacid molecule is in concentration excess compared to said target nucleicacid.
 53. The method of claim 31, wherein a plurality of said secondnucleic acid molecule is provided, such that said second nucleic acidmolecule is in concentration excess compared to said target nucleicacid.
 54. The method of claim 31, wherein said target nucleic acid andsaid second nucleic acid molecule form a duplex, and wherein a pluralityof said first nucleic acid molecule is provided such that said firstnucleic acid molecule is in concentration excess compared to saidduplex.
 55. The method of claim 54, wherein said cleaving said cleavagestructure comprises cleaving said first nucleic acid molecule togenerate non-target cleavage product.
 56. The method of claim 55,wherein said non-target cleavage product from said first nucleic acidmolecule is generated in concentration excess compared to said duplex.57. The method of claim 31, further comprising providing a third nucleicacid molecule complementary to a third portion of said target nucleicacid upstream of said first portion of said first target nucleic acid,wherein said cleavage structure comprises said third nucleic acidmolecule.